REESE LIBRARY
UNIVERSITY OF CALIFORNIA.
•.••>-;-. . -;.-
1
HANDBOOK
FOR
BIO-CHEMICAL LABORATORY.
INCLUDING
METHODS OF PREPAEATION
AND
NUMEROUS TESTS
ARRANGED ALPHABETICALLY.
BY
JOHN A. MANDEL,
\\
Professor of Chemistry at the New York College of Veterinary Surgeons,
and Assisfant to the Chair of Chemistry, etc., at the Bellevue
Hospital Medical College and the College of
the City of New York.
FIRST EDITION.
FIRST THOUSAND.
NEW YORK:
JOHN WILEY & SONS.
LONDON : CHAPMAN & HALL, LIMITED.
1896.
OP 5/7
Copyright, 1896,
BY
JOHN A. MAN DEL.
PEEFACE.
IN this little handbook an attempt has been made to give
concise directions for preparing the most important sab-
stances that enter into the composition of the fluids and
tissues of the animal body. The methods herein presented
are compiled from the most recent and important works on
physiological chemistry; and in certain instances two or three
procedures are given for obtaining the same result.
The two hundred or more tests are arranged in alphabetical
order ; and the name of the scientist who suggested the test,
or the name under which it is ordinarily known, is given in
each case. My most earnest desire in compiling this hand-
book has been both to facilitate general work in bio-chemical
laboratories and to afford the student an opportunity to have
conveniently at hand all the necessary facts in modern
scientific testing, so that loss of time in consulting works of
reference might be reduced to a minimum.
A. HANDEL.
COLLEGE OF THE CITY OF NEW YORK,
January, 1896.
ill
LIST OF PREPARATIONS,
PAGE
Adenin 55
Albuuiiuates 14
Allantoin 51
Ainylopsin 36
Artificial gastric juice ........ 32
Aspartic acid 40
Bilirubiii 28
Biliverdin 29
Carbon monoxide haemoglobin . 21
Casein 13
Cholalic acid 25
Cholesterin 30
Creatin 53
Creatinin , . 54
Dextrose 4
Fatty acids 7
Fibrin , 15
Fibrin ferment 22
Fibrinogen 11
Glucose 4
Glutamic acid 41
Glycocholic acid 23
Glycocoll 26
Glycogen 1
Glycuronic acid 60
Guanin 55
Haemoglobin 21
Hippuric acid 50
Hypoxanthin 55
Indol 41
Inosit 6
Lactic acid (fermentation) 44
PAGE
Lactic acid (sarco) 45
Lactose 2
Lecithin 19
Leucin 37
Maltose 3
Methaemoglobin 22
Myosin 11
Nuclein 17
Nucleinic acids 18
Ov-albumin 9
Ov-vitelliu 12
Oxyhaemoglobin 20
Pepsin 31
Peptones 16
Protagon 46
Ptyalin 81
Renuin 34
Serum albumin 8
Serum globulin 10
Skatol 42
Soap 8
Steapsin 37
Taurin 27
Taurocholic acid 24
Trypsin 35
Tyrosin 37
Urea 47
Uric acid 48
Urobilin... 58
Vitellin.., 12
Xanthin 55
HANDBOOK FOR BIO-CHEMICAL
LABORATORY.
Olycogen, C6H1005.
Preparation. — 1. Kill a large well-fed rabbit by cutting its
throat, open the abdomen immediately and remove the liver.
After weighing, cut it up in rather large pieces and quickly
throw them in boiling water (about 400 c.c. to 100 grms. liver),
and let boil for half an hour. Then remove the pieces and
grind them up finely in a mortar, return to the boiling water
and add caustic potash solution (3-4 grms. KOH to 100 grms.
liver). Now warm on the water-bath, and allow it to concen-
trate until you have 200 c.c. of liquid for every 100 grms. liver.
If a scum forms on the surface, place the liquid in a beaker,
covering it with a watch-glass, and heat it until all has dis-
solved, then put aside to cool. Neutralize with HC1 and pre-
cipitate the albuminous bodies by the alternate addition of HC1
and a solution of potassio-mercuric iodide * ^Brilcke reagent)
in small portions. The addition of potassiovmercuric iodide
must be continued until no further precipitate occurs. If
the liquid at last remains milky, nearly neutralize with
caustic soda, and then treat with HC1 again. Filter off the
precipitate of albuminous bodies through thick filter-paper,
and wash by removing the precipitate from the filter by means
of a spatula and place it with water containing HC1 and
* This solution is prepared by saturating a boiling, not too concen-
trated (10*) solution of potassium iodide with pure mercuric iodide and
filtering after cooling.
2 HANDBOOK FOE BIO-CHEMICAL LABORATORY.
potassio-mercuric iodide and then return to filter. This
treatment must be repeated at least four times. The
several filtrates are united and, while stirring, treated with 2
vols. 96$ alcohol, which precipitates the glycogen, and allow
it to stand in a cool place overnight. Filter off the precipitate
and wash first with 62$ and then with 98$ alcohol. This
glycogen generally contains but a trace of albumins, but if re-
quired more pure, dissolve it while still moist in a little warm
water, add some HC1 and potassio-mercuric iodide after al-
lowing to cool, and proceed as above. Lastly, wash the glyco-
gen, which has been previously treated with absolute alcohol,
a couple times with ether, and allow it to dry in the air or
over sulphuric acid. (It. Eulz.}
2. Briiclce's method consists in precipitating the albumin-
ous bodies from the watery extracts by HC1 and potassio-
mercuric iodide without previously extracting with caustic
potash, and then proceeding as above directed.
Properties. — Glycogen is a white amorphous powder, easily
soluble in hot water, yielding an opalescent solution, which
when allowed to evaporate on the water-bath forms a pellicle
over the surface which disappears again on cooling. The
solution is dextro-rotatory, (a) D = -j- 211° (Kulz). On boil-
ing with dilute mineral acids, or by the action of diastatic
enzymes (ptyalin, diastase), it is readily converted into malt-
ose, isomaltose, and dextrose. Its solution does not reduce
Fehling's solution on boiling, but holds copper oxyhydrate in
solution in alkaline liquids. With a solution of iodine glyco-
gen solutions are colored wine-red which disappears on heat-
ing. Glycogendoes not ferment with yeast.
Lactose, C12H22On+H20.
(MILK-SUGAR.)
Preparation. — The sweet whey obtained after the precipi-
tation of casein from milk (see page 13) is heated to boiling,
HANDBOOK FOR BIO-CHEMICAL LABORATORY.
filtered, evaporated to dryness with magnesium carbonate, and
the residue extracted with alcohol. Exhaust the part insol-
uble therein with hot water, filter, and evaporate the filtrate
to a syrupy consistency, and allow to stand in a cool place un-
til the lactose crystallizes out. If the syrup is at all colored,
the solution must be decolorized by passing the solution
through animal charcoal.
Properties. — Lactose crystallizes in rhombic prisms which
contain a molecule of water of crystallization. It is soluble
in 6 parts cold and 2.5 parts hot water. It has only a faint
s \veut taste. It is insoluble in ether or absolute alcohol.
Aqueous solutions are dextro-rotatory, (a) D — -f- 52.5°.
Milk-sugar combines with bases ; the alkali combinations are
insoluble in alcohol. Solutions of lactose reduce Fehling's
solution, but less powerfully than dextrose. On warming
with phenyl-hydrazine acetate it gives on cooling a yellow
precipitate of phenyl-lactosazon, C24H32N409. By boiling
with water, or more readily on boiling with acids, or by
means of inverting ferments, as in the alimentary canal, it
takes up water and is converted into a glucose called galac-
tose. It undergoes alcoholic fermentation by the action of
certain schizomycetes, producing lactic acid at the same time.
Maltose, C12H,20U+ H30.
Preparation. — 500 grms. potato-starch are thoroughly
mixed with 2.5 litres cold water and converted to a paste by
heating on the water-bath. Allow this paste to cool to
60-65° C., and add a watery extract of 30-35 grms. of air-dried
malt made at 40° C. Keep at 60° C. for an hour, then boil,
filter, and evaporate to syrup in a flat porcelain dish. This
is extracted several times with boiling 90$ alcohol. If no
crystals of pure maltose are at your disposal, boil a portion of
the syrup with absolute alcohol, filter, evaporate to thin syrup,
and allow it to stand in thill layers until it crystallizes, which
4 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
generally takes place in a few days. In the meantime distil
most of the alcohol off from the main portion, evaporate the
residue to a thick syrup, and on cooling stir into this a few
crystals of the pure crystallized maltose. After three to five
days the syrup will have crystallized to a stiff mass of crystals.
These are rubbed to a thin paste with methyl alcohol,
drained on paper, and washed once with methyl alcohol,
pressed, washed again with methyl alcohol, and purified by
further crystallization. For this purpose dissolve 500 grms.
of the dried, pressed maltose in 15 c.c. water on the water-
bath, add 130 c.c. 90$ alcohol, boil, filter, and allow to cool.
No syrup should separate out. Add a few crystals of pure
maltose, and shake often, until the entire liquid after a few
hours crystallizes into a thick mass of crystals. After draining
the crystals they may be recrystallized from methyl alcohol,
which is done by heating 50 grms. of the crystals with 12 c.c.
water until all has dissolved, and adding 300 c.c. methyl alco-
hol; boil, filter, and allow to cool. Shaking facilitates crystal-
lization considerably. (Soxlilet.)
Properties. — Maltose crystallizes generally in microscopic
needles containing 5$ (1 mol.) water of crystallization. The
dried crystals are hygroscopic, specific rotatory power being
(«) J}= -J- 137°. Maltose reduces alkaline solutions of copper,
bismuth, and other metallic salts, but its reducing power as
measured by Fehling's solution is -| less than that of dextrose.
With phenyl-hydrazine acetate it gives after heating for 1-J
hours clusters of yellow crystals, C24H32N409, melting at 206° 0.
Maltose is easily and completely fermented by yeast. When
heated with very dilute sulphuric acid, maltose yields dex-
trose. The diastatic enzymes act in the same way.
Dextrose, C6H12Ofi.
Preparation. — Warm a mixture of 1.5 litres 90^ alcohol
and 60 c.c. strong HC1 on the water-bath to 45° C., and
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 5
gradually add 500 grms. powdered cane-sugar, stirring all
the while and taking care that the temperature does not
rise above 50° C. After two hours the sugar will have dis-
solved and will be inverted into dextrose and laevulose ; allow
it to cool, and place it in a cold place until crystallization com-
mences, which occurs in from six to eight days. The crystal-
lization may be facilitated by constant stirring. If pure anhy-
drous crystals of glucose are at hand, add a few grammes to
the cold solution and stir well. In this case the crystalliza-
tion will begin in a few hours, and is complete in 36 hours.
The crystals thus obtained are well drained, then washed free
from HC1 by 90$ alcohol, then with absolute alcohol, and
now dried at a moderate heat. To completely purify these
crystals boil them for five to ten minutes with pure methyl
alcohol* (sp. gr. 0.810 at 20° C.), filter, quickly cool, and the
glucose crystals will separate out. (Soxhlet).
Properties. — Dextrose (glucose, grape sugar) is readily sol-
uble in water, sparingly soluble in alcohol, and insoluble in
ether. It crystallizes from an aqueous solution in white
spheroidal masses, and from alcohol in transparent anhydrous
prisms. Its solutions rotate the ray of polarized light to the
right : (a) D — -f- 52.6°. In alkaline solutions dextrose
reduces salts of silver, bismuth, mercury, and copper. Under
the influence of yeast it is converted into alcohol and carbon
dioxide. It may also undergo lactic-acid fermentation under
the influence of certain bacterial growths. With a mixture
of 2 parts phenyl-hydrazine hydrochloride and 3 parts sodium
acetate a watery solution of glucose gives, when heated on
the water-bath, a precipitate of fine yellow needles (phenyl
glucosazon, C18H21N404), melting at 204°-205° C.
* The purest methyl alcohol is mixed withjabout 20# water, and about
four-fifths distilled off on the water-bath. This distillate has, as a
rule, the above specific gravity, and is immediately used.
6 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
Inosit,, C6H1206.
preparation. — 1. Make a watery extract of 2 Ibs. chopped
meat, remove the albuminous bodies by coagulating at boil-
ing heat. This is filtered and the filtrate precipitated by
sugar of lead, and again filtered and washed. This filtrate is
boiled with basic lead acetate and allowed to stand 24-48
hours. The precipitate thus obtained, which contains all the
inosit, is decomposed in water by H2S. The filtrate is strongly
concentrated, treated with 2-4 vols. hot alcohol, and the
liquid removed as soon as possible from the tough and flaky
masses which ordinarily separate. If no crystals separate
from the liquid within 24 hours, then treat with ether until
the liquid has a milky appearance and allow it to stand. In
the presence of a sufficient quantity of ether, crystals of inosit
separate within 24 hours. The crystals thus obtained, as also
those which are obtained from the alcoholic solution directly,
are recrystallized by redissolviug them in very little water
and the addition of 3-4 vols. alcohol.
2. Inosit may also be prepared from green beans by evap-
orating the watery extract to a syrupy consistency and pre-
cipitating with alcohol. The precipitate is dissolved in water
and the inosit allowed to crystallize out. (Void.}
Properties. — Inosit crystallizes in large, colorless, rhombic
crystals of the monoclinic system, or, if not pure and if only
a small quantity crystallizes, it forms fine crystals similar to
cauliflower. The crystals melt at 217° 0. It dissolves in 6
parts water at the ordinary temperature, and the solution has
a sweetish taste. It is insoluble in strong alcohol and in
ether. Inosit does not ferment with beer yeast, but is cap-
able of lactic-acid fermentation. It dissolves copper oxhy-
drate in alkaline solutions, but does not reduce on boiling.
It gives negative results with MOORE'S or BOETGER-ALMEN'S
bismuth test. Its solutions have no action on polarized light.
Inosit gives no combination with phenyl-hydrazin acetate.
HANDBOOK FOR BIO-CHEMICAL LABOKATOEY. 7
Fatty Acids, CnH2n02.
Preparation. — Dissolve 20 grms. caustic potash in 100 c.c.
absolute alcohol, placing the vessel in cold water as consider-
able heat is generated. When all has dissolved that will,
decant the clear solution from the sediment. Now heat on
water-bath 50 grms. mutton tallow or leaf lard with 50 c.c.
alcohol in a flas~k connected with a return condenser. Continue
the application of heat until all the fat has melted; now add
the potash solution, and gently boil for one half to one hour.
"When the liquid in the flask does not give a cloudiness when
added to water, then all the fat has been converted into soap.
Filter through a cotton plug and dilute this liquid with 500 to
600 c.c. water* Boil over the naked flame until all the odor of
alcohol has disappeared, and add dilute sulphuric acid (1 to 4)
until the solution has a marked acid reaction. Allow this to
stand on the boiling water-bath until the separated fatty
acids have collected on the surface as an oily layer. Now
allow to cool, filter through a wet filter, wash the fatty acids
with cold water, and crystallize the same from hot 80$ alco-
hol. The oleic acid (C18H3402) remains nearly entirely in the
mother liquid, while the palmitic (C]eH32OJ and stearic acid
(C18H3602) forms the crystals. Determine the melting point
of the mixture, then dissolve the same in cold alcohol, and
fractionally precipitate this solution with an alcoholic solu-
tion of sugar of lead (3 to 4 fractions are sufficient). Each
precipitate is shaken with ether and the fatty acid obtained
on the evaporation of the ether. Determine the melting-
point of each fraction, and a different melting-point will be
found for each, showing that the fatty acids obtained from
the fat consists of a mixture. The first lead precipitate con-
tains the stearic acid.
Stearic acid melts at 69.2° C.
Palmitic acid melts at 62° C.
8 HANDBOOK FOE BIO-CHEMICAL LABOEATOEY.
Soap.
Preparation. — Dissolve 50 grms. fatty acids (page 7) in
100 c.c. alcohol by warming on the water-bath. Gradually
add an alcoholic solution of caustic soda (10 grms. NaHO in
100 c.c. alcohol) to this solution until a very faint alkaline
reaction is obtained. Heat on water-bath for 15 minutes,
transfer to flat porcelain dish, and evaporate off the alcohol
on the water-bath. When nearly all alcohol is off, add 30 c.c.
water and continue the evaporation, stirring all the while.
The product thus obtained when drv will be a neutral soap.
Serum Albumin.
Preparation. — 1. Defibrinated ox blood (or human transu-
dations) is filtered through washed linen (free from starch)
and allowed to stand in the cold in a tall vessel until the red
blood-corpuscles have settled to the bottom. The clear serum
is carefully drawn off by means of a siphon and saturated at
30° C. with magnesium sulphate, filtered at the same tem-
perature, and washed with a saturated solution of magnesium
sulphate. Saturate the filtrate with sodium sulphate (or
ammonium sulphate) at 40° C., whereby the serum albumin
is precipitated. This precipitate is collected on a filter,
pressed between paper, dissolved in water, reprecipitated by
sodium sulphate (or ammonium sulphate), and the process
repeated several times. The solution in water is now freed
from salts by means of dialysis, using large amounts of dis-
tilled water. The serum albumin may be obtained from this
dialyzed solution by evaporating the solution to dryness at a
gentle heat, or, better, by precipitating with an excess of
strong alcohol, filtering, washing with alcohol, and finally
with ether, and then drying by exposure to the air.
When precipitating the serum albumin by means of alcohol
filter immediately, press between paper, and remove the alco-
hol from tte precipitate by means of ether.
HANDBOOK FOR BIO-CHEMICAL LABORATORY.
2. Serum albumin may also be precipitated from the fil-
trate, after the precipitation of the serum globulin, by means
of acetic acid — about 1$. Filter after a few hours, press the
precipitate between filter-paper, dissolve in water, neutralize
by the addition of alkali, and remove salts by means of dial-
ysis. The serum albumin is obtained from this salt-free solu-
tion as above directed.
Properties. — In the dry state serum albumin forms a trans-
parent, gummy, brittle, hygroscopic mass or a white powder,
readily soluble in water, forming a clear solution with a spe-
cific rotatory power, for a solution saturated with JSTaCl, of
(a) D = — 62.6° to 64.6°. The coagulation temperature is
+ 70° to 75° C., but varies with the varying concentration
and the amount of salts. Its solutions are precipitated by
alcohol and ether.
Oy-Albumin.
Preparation. — 1. The white of several hen's eggs (free
from yolk) is subdivided by cutting with a scissors or by
beating violently, filtered through linen (free from starch),
and then treated with an equal amount of water. A precipi-
tate will form, and this must be removed by filtration. Sat-
urate the filtrate at 20° C. with very finely powdered mag-
nesium sulphate (MgS04 -f- 7HaO), which is done by adding
small portions of the salt at a time and constantly stirring.
After completely saturating, remove the precipitated globu-
lins by filtration, and thoroughly dialyze the filtrate until a
portion removed and treated with BaCl2 does not give any
reaction for sulphates. Evaporate the solution (which greatly
increases in volume during dialysis) at 40°-50° C. in a flat
dish, and allow this concentrated solution to undergo dialysis
again ; and, lastly, evaporate to dryness at the above-mentioned
temperature.
2. Ov-albumin may also be obtained by saturating the fil-
10 HANDBOOK FOR BIO CHEMICAL LABORATORY.
trate from the magnesium sulphate with sodium sulphate
(or ammonium sulphate) at 20° C. The precipitate of ov-
albumin is filtered off and pressed between filter-paper, dis-
solved in water, again precipitated with sodium sulphate (or
ammonium sulphate), and after repeating this process several
times the salts are removed by dialysis, and the salt-free
solution evaporated to dryness at 40° C., or in a vacuum.
Properties. — The ov-albumin remains as a yellowish, trans-
parent mass soluble in water, precipitated from its solutions
by alcohol, and quickly converted into coagulable albumin. It
is not precipitated from its watery solution by MgS04, but
completely precipitated by NH4S04. Its 1-3$ solution con-
taining some salt coagulates at about 56° C. It is not pre-
cipitated by ether, and has a specific rotatory power of (a)
V = —35.5°.
Serum Globulin or Paraglobulin.
1. Faintly acidify blood serum (prepared as directed on
page 8) with a few drops acetic acid, and dilute with 10-20
vols. of water. The serum globulin will separate as a fine
flocculent precipitate, which is filtered and further purified
by dissolving it in a dilute common-salt solution or in water
by the aid of the smallest possible amount of alkali, and then
reprecipitating by diluting with water or by the addition of a
little acetic acid. On repeating this twice the serum globulin
is carefully dried in the air. (Al. Schmidt.)
2. Serum may also be precipitated from blood serum by
means of magnesium or ammonium sulphate added to satu-
ration. Filter and wash with one-half saturated solution of
the salt used, and purify the precipitate by means of dialysis.
As ammonium sulphate is removed by dialysis with difficulty,
it is best to use magnesium sulphate. When great purity is
required the precipitate may be redissolved by adding distilled
water, reprecipitating by saturating with the salt again, and
purifying this by thorough dialysis. (Hammarsten.)
HANDBOOK FOR
Properties. — Serum globulin is insoluble in water, but
soluble in dilute salt solutions (NaCl, (NH4)2S04, MgSOJ.
Its solution in dilute solutions of (KH4)J304, or MgS04, are
completely precipitated by saturating these solutions with the
respective salt, but it is incompletely precipitated by NaCl.
The coagulation temperature with 5-10$ NaCl in solution is
75° C. Specific rotatory power for a solution containing salt
is (a) D = - 47.8.
Fibrinogen.
Preparation. — Precipitate salt plasma with an equal volume
of a saturated solution of NaCl (33$). The precipitate thus
obtained is pressed between filter-paper, redissolved in an 8$
salt solution, the filtrate precipitated by a saturated salt solu-
tion as above, and after precipitating in this way three times,
the precipitate at last obtained is filtered, pressed between
filter-paper, and finely divided in water. These operations
should be performed rapidly, as prolonged contact with a half-
saturated salt solution renders the precipitate of fibrinogen
very insoluble. The fibrinogen dissolves by the aid of the
small amount of NaCl contained in itself, and the solution
may be made salt free by dialysis with very faintly alkaline
water. (Hammarsten.)
Properties. — Fibrinogen has the general properties of the
globulins, namely, insoluble in water, but soluble in dilute
neutral salt solutions. It is precipitated unchanged from
these solutions on sufficiently diluting with water. On heat-
ing it coagulates. When dissolved in a 5-10$ NaCl solution it
coagulates at 52° to 55° C , and the faintly alkaline or nearly
neutral weak solution coagulates at 56 C. Its specific rota-
tory power for sodium light is — 52.5°.
Myosin,
Preparation. — 1. Finely chopped meat is extracted by 5$
magnesium-sulphate solution. The filtered extract is then
12 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
treated with MgS04 in substance until 100 c.c. of the liquid
contains about 50 grms. of the salt. The so-called paramyosin
or musculin separates. The filtered liquid is now treated
with magnesium sulphate until each 100 c.c. of the liquid
holds 94 grms. MgS04 in solution. The myosin which now
separates is filtered, dissolved in water by the aid of the re-
tained salt, precipitated by diluting with water, and, when
necessary, purified by redissolving in dilute salt solution and
precipitating with water. (Halliburton.)
2. Myosin may also be prepared by treating finely chopped
meat, which has first been soaked in cold water until the
muscles are white, with a 10-20$ ammonium-chloride solu-
tion, allowing to stand for a few hours, stirring now and then.
Filter this solution off and dilute with about 20 vols. water.
The myosin separates in flakes which gradually settle to the
bottom. This is washed 3 to 4 times by decantation, but not
oftener, as then the myosin becomes insoluble. Dissolve now
in ammonium-chloride solution and the myosin obtained
therefrom, either by reprecipitating by diluting with water, or
by removing the salt by dialysis. (DanileivsJcy.)
Properties. — Myosin has the general properties of the
globulins. It is completely precipitated by saturating with
NaCl, also by MgS04, in a solution containing 94$ of the salt
with its water of crystallization. Like fibrinogen it coagulates
at 56° C. in a solution containing common salt, though the
coagulation temperature may vary for myosins of different
origin, and also for the same myosin in different salt solutions.
It is soluble in dilute alkalies. Myosin decomposes hydrogen
peroxide.
Ovo-Yitellin.
Preparation. — Shake the yolks of two eggs with 200 c.c. acid-
free ether in a stoppered cylinder, then add 5 c.c. alcohol. A
sticky, stringy precipitate will be formed. Remove the ether
as well as possible and add 100 c.c. of a 10$ common salt
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 13
solution. On shaking the precipitate it dissolves in the com-
mon salt solution, yielding a cloudy liquid ; place the solution
in a separatory funnel and shake with an equal volume of
ether. It will remain clear or nearly so. Draw off the
watery solution and allow it to stand until the next day,
when a cloudiness will have appeared; this is settled byre-
shaking again with ether. Draw off the watery liquid again,
measure it, and dilute with 10 vols. of water. The very fine
precipitate produced is filtered off the following day, washed
with water and then with alcohol. To further purify the
substance thus obtained place the precipitate in a flask, boil
with absolute alcohol on the water-bath, filter, wash with alco-
hol, then with ether, and lastly subdivide the mass in a flat
dish and allow to dry over sulphuric acid or in a vacuum.
(Salkowski.)
Properties. — Vitellin is insoluble in water, but soluble in
dilute neutral salt solutions. It is soluble in hydrochloric acid
of 1 p.m. and in very dilute solutions of alkalies or alkali
carbonates. The coagulation temperature for the solution
containing NaCl lies between 70° and 75° C. It yields nuclein
when digested with pepsin and hydrochloric acid. Vitellin
obtained as above should contain only 0.95$ phosphorus.
Casein.
Preparation. — 200 c.c. fresh milk are diluted with 800 c.c.
water and treated with acetic acid so that the dilute milk con-
tains 0.75-1 p.m. acetic acid. (Hydrocloric acid may also be
used.) The casein hereby precipitated is quickly washed with
water by decantation and rubbed with water in a mortar so that
it is as fine as possible. Dissolve it with the least possible quan-
tity of a 0.1$ caustic-soda or ammonia solution, continually
stirring, and taking care that the liquid does not became alka-
line but neutral. The milk-white liquid is filtered through
several folds of filter-paper, when it will become water-clear
14 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
with only a slight bluish opalescence. After diluting with
water it is again precipitated by acetic acid (or hydrochloric
acid) as above directed, and the precipitate again ground
finely, washed on a filter with water, and then dissolved in caus-
tic soda as above. This is repeated once or twice. Then the
washed precipitate is gently pressed, quickly rubbed to a
paste with 97$ alcohol, transferred to a filter, washed first
with alcohol, then with ether, pressed, then dried in a mortar
after having finely divided it. The last traces of ether are
removed in a vacuum or over sulphuric acid. (Hammarsten.)
Properties. — Casein forms a white, dusty, insoluble powder
which reddens moist blue litmus-paper. It is readily soluble
in dilute alkalies and acids. It is completely soluble in 0.2$
hydrochloric acid, and if this is digested at 38-40° 0. with
pepsin a gradual cloudiness is formed and a precipitate of
nuclein is produced. Casein solutions do not coagulate on
boiling, but are covered, as milk, with a skin. Casein is pre-
cipitated from neutral solutions or from milk by NaCl or
MgS04 in substance without changing its properties. Metallic
salts, such as copper sulphate, completely precipitate casein
from neutral solutions. Casein coagulates with rennet or
chymosin in the presence of lime-salts.
Albuminate (Alkali).
Preparation. — Beat up the white of an egg finely and filter
through a piece of clean linen, and treat the filtrate with
a solution of 1 grm. caustic potash in a little water, contin-
ually stirring. The solution will be immediately, or after
some time, converted into a gelatinous mass. This is cut into
pieces, washed a few times with water, then dissolved in warm
water, allowed to cool, and precipitated by acetic acid, wash-
ing the precipitate with alcohol and ether. This precipitate
of alkali albuminate appears as a flaky, amorphous, white
substance, nearly insoluble in water as well as NaCl solutions,
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 15
but readily soluble in alkalies Xa3C02, Na2HP04 , as well as
dilute hydrochloric acid. It does not coalgulate on applying
heat to its solutions.
Albuminate (Acid).
Preparation. — Digest the white of two eggs with hydro-
chloric acid (0.5$), and let it stand or apply gentle heat ; then
dilute with twice its volume of water. Collect the precipi-
tate, dissolve it in hot water, and carefully neutralize the solu-
tion with Na2C03; finally wash well with water, and the prod-
uct will be pure acid albuininate.
Fibrin,
Preparation. — Whip freshly-drawn ox-blood with a bunch
of twigs ; the fibrin adheres to the twigs and entangles but a
few blood-corpuscles. The mass is washed for a long time in
a stream of ruuning water until nearly white, and then with a
5$ common salt solution, and again with water. When free
from NaCl extract with alcohol and then with ether, and pre-
serve in a solution of equal parts glycerine and water.
Properties. — Fibrin is soluble with difficulty in a 5-10$
common salt or -saltpetre solution or similar solutions of
MgS04 or other neutral salts. In the presence of enzymes or
by putrefaction it may dissolve. It is insoluble in water, alco-
hol, or ether. Fibrin decomposes hydrogen peroxide. Solu-
tions of fibrin are precipitated by lead acetate, copper sulphate,
and mercuric chloride. Weak HOI (0.2$) causes fibrin to
swell up into a transparent jelly, while stronger acids dissolve
it in a time with the formation of acid albumin or syntonin
and albumoses.
16 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
Peptone,
Preparation.—!. Digest 2000 grms. washed fibrin, but not
boiled, with 5 litres of a solution of purified pepsin (see Pep-
sin) obtained from 600 grms. of the removed mucous mem-
brane of the fundus of the stomach of two pigs and contain-
ing 0.4$ HC1. This mixture is allowed to digest for a fort-
night at 37°-40°C., so as to insure as complete a conversion of
the albumoses into peptones as possible. A little thymol
(0.25$) may be added to prevent putrefaction. The forma-
tion of peptones takes place much more quickly with trypsin
digestion ; therefore it may be used instead of pepsin. After
complete digestion the solution is filtered through linen and
should not contain any albuminates or coagulable albumin.
Exactly neutralize the filtrate with soda and heat to boiling.
While boiling hot precipitate by saturating with ammonium
sulphate. Allow to cool, and separate the precipitated albu-
moses and the ammonium sulphate, which has crystallized
out, by filtration. The filtrate is again heated to boiling,
and made strongly alkaline with ammonia and ammonium
carbonate and saturated with ammonium sulphate while boil-
ing. Filter when cold, and boil the filtrate until the odor of
ammonia has entirely passed off ; now saturate again with
ammonium sulphate while hot, and acidify with acetic acid.
When cold filter and strongly concentrate the filtrate, stirring
constantly, and when cold decant the liquid from the salts which
have crystallized out. A great part of the remaining ammon-
ium sulphate may be removed by careful fractional precipitation
with alcohol (£ vol.), so that at last a solution rich in peptones,
containing alcohol and some little ammonium sulphate, is ob-
tained. The alcohol is driven off by boiling the solution,
and the ammonium sulphate decomposed by boiling with
barium carbonate. The filtrate is freed from the excess of
barium by the careful addition of dilute sulphuric acid. This
last filtrate, which must not contain any sulphuric acid, is
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 17
strongly concentrated, and the peptones precipitated by the
addition of alcohol. The peptones thus obtained are quickly
dried in a desiccator connected with an air-pump. (KilJine.)
2. The solution obtained after the digestion of fibrin with
pepsin as described in method 1 is first neutralized with
sodium hydrate, filtered through linen, and the filtrate acidi-
fied with acetic acid and concentrated considerably. Now
precipitate the albumoses by saturating with amonium sul-
phate, filter and press, and boil the solution with barium hy-
drate, and finally with barium carbonate and a large quantity
of water, until ammonia can no longer be detected. Eemove
the barium sulphate by filtration through cloth bags, and the
filtrate evaporated after washing the precipitate. The barium
peptone thus obtained is decomposed by a slight excess of
sulphuric acid, the barium sulphate filtered off, the solution
concentrated, the free acid neutralized with ammonia, and
after cooling add 6$ sulphuric acid (previously diluted).
Now precipitate with a large excess of phospho-tungstic acid,
filter, wash first with 6$ sulphuric acid, then with a large
quantity of water, after which the compound is decomposed
by means of barium hydrate and the excess of barium com-
pletely removed from the filtrate by the careful addition of
sulphuric acid. The solution of peptones thus obtained has a
distinct acid reaction, and the solid peptone may be obtained
therefrom by repeated precipitation and boiling with alcohol.
(Kilhne and Cliittenden.)
Nuclein.
Preparation. — 1. Pure casein is dissolved in water contain-
ing 2 p.m. HC1 and filtered, and the filtrate digested with pep-
sin at 40° C. After a time a precipitate of nuclein appears; this
is filtered off, washed with warm water, and purified by re-
peated solution in a \% solution of sodium carbonate and re-
precipitated after filtration by the addition of dilute hydro-
18 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
chloric acid. The nuclein thus obtained is washed with
water and extracted with alcohol and then ether. Dry over
sulphuric acid.
2. Suspend some beer-yeast in water and wash by decanta-
tion, and then add it to water containing 5 p.m. HC1. After
some time add a slight excess of soda. Filter immediately
through a rapid filter and allow to flow into dilute HC1, when
a precipitate will fall to the bottom of the flask. Wash by
decantation first with dilute hydrochloric acid, then with
water, and then with boiling alcohol. Dry in a vacuum.
Properties. — The nucleins are colorless, amorphous, insol-
uble or very slightly soluble in water. They are insoluble in
alcohol and ether. They are more or less readily soluble in
alkalies ; in dilute mineral acids they are insoluble or dissolve
with difficulty. On boiling with caustic alkali they decom-
pose and alkali phosphates are formed. On fusing with soda
and saltpetre they give alkali phosphates also.
Nucleinic Acids.
Preparation. — Treat 1000 c.c. well-washed yeast with 3250
c.c. of a 3$ caustic-soda solution, and allow to digest at the
temperature of the room for 5 minutes. Neutralize with hy-
drochloric acid, and add an excess of acetic acid. Filter off
the precipitated albuminous bodies and measure the filtrate.
Now add HC1 to filtrate so that it contains 3-5 p.m., and
then add an equal volume alcohol which has previously been
acidified to the same extent (3-5 p.m.). The impure nu-
cleinic acid thus precipitated is filtered off, dissolved in am-
moniacal water, and purified by treating as above, namely,
acetic acid, hydrochloric acid, and alcohol. The purified
product is dried in desiccator. (Altmann.)
Properties. — Nucleinic acids are white, amorphous, and
acid in reaction, readily soluble in ammoniacal or alkaline
water, and are not precipitated therefrom by an excess of
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 19
acetic acid, but readily by a slight excess of hydrochloric acid,
especially in the presence of alcohol, They are insoluble in
alcohol and ether.
Lecithin.
Preparation. — The yolk of several eggs free from white are
violently shaken with ether until the ether does not dissolve
any more. The several etheral extracts are freed from ether
by distillation, and the residue dissolved in petroleum ether
and filtered. The filtrate is transferred to a separatory funnel
and shaken several times with 75$ alcohol; the alcoholic ex-
tracts are united, and allowed to stand until entirely clear.
Separate any petroleum ether present, and filter. The last
portions of petroleum ether are distilled off, and the residue
is allowed to stand in a cold place for several days, when a
precipitate will be produced. The liquid is separated from the
precipitate by decantation and then filtration. Decolorize the
solution by boiling with animal charcoal, filter, and evaporate
to syrupy consistency at 50-60° 0. Dissolve the syrup in
ether, filter, and evaporate the filtrate, when the lecithin will
be obtained in a nearly pure form. To further purify, dis-
solve in as little absolute alcohol as possible, and on cooling
this solution in a freezing mixture to —5° to — 15° C. the
lecithin will be deposited. This is filtered while cold, and
dried in a vacuum over sulphuric acid. (E. Gilson.)
Properties. — Lecithin is a soft, waxy, mouldable, not mark-
edly crystalline mass, which when placed on a microscope slide
with water forms oily drops, appearing under the microscope
like worms, called "myeliue forms." It is easily decom-
posed by acids and alkalies, especially by the latter. On
fusion with soda and saltpetre on platinum foil it yields
a mass in which phosphoric acid can be easily detected. It
combines with acids and bases. The combination with HC1
gives a double salt with PtCl4, which is insoluble in alcohol,
20 HANDBOOK FOB BIO-CHEMICAL LABORATORY.
but soluble in ether, and which contains 10.2$ platinum. It
is soluble in chloroform, carbon disulphide, benzol, and fatty
oils.
Oxyhaemoglobin.
Preparation. — Defibrinated blood (best from the dog or the
horse) is mixed with at least 10 times its volume of a salt solu-
tion (1 vol. saturated solution with 9 vols. water), and allowed
to stand in a cool place for a couple of days until all the blood-
corpuscles have settled to the bottom. Remove the superna-
tant liquid by means of a siphon, and transfer the blood-cor-
puscles to a flask. Add 2 vols. water, and then shake with an
equal volume of ether. After a time remove the ether by de-
cantation, and allow the ether retained by the blood solution
to evaporate in an open dish in the air. Filter quickly
through a folded filter, cool the solution to 0° C., and add while
stirring J of its volume of alcohol which has been cooled to
0° C. and allow to stand for several days at —5° to —10° C.
The crystals which separate are filtered off, pressed, and re-
crystallized by first quickly dissolving them in not too much
water at 20° to 30° C., cooling to 0° C., precipitating with cold
alcohol as above, and allowed to stand at —5° to — 10° C. This
recrystallization is repeated several times. Lastly, the crystals
are washed with cooled water containing alcohol (£ vol. alco-
hol), and dried in vacuum at 0° 0. or a lower temperature.
(Hoppe Seyler.)
Properties. — Oxyhaemoglobin from various kinds of blood
has different crystalline forms. It is insoluble in ether, chlo-
roform, benzol, and carbon disulphide. Crystals of oxyhaemo-
globin are insoluble in absolute alcohol. Its solution in water
is not precipitated by many metallic salts, but is precipitated
by sugar of lead. On heating the watery solution it decom-
poses at 60°-70° C., and splits into hasmatin and albumin. It
is also decomposed by acids, alkalies, and many metallic salts.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 21
Dilute solutions show a spectrum with two absorption-bands
between the Fraunhofer lines D and E. The one band, a,
which is narrower but darker and sharper, lies on the line D\
the other, broader, less defined and less dark band, ft, lies at
E. Oxyhaemoglobin solutions are reduced by reducing solu-
tions such as ammonium sulphide, ammoniacal ferro-tartrate
solution (Stokes reduction liquid) yielding a characteristic
spectrum of haemoglobin.
Haemoglobin.
•
Preparation. — Fill a cylinder nearly full with a dilute
watery solution of oxyhaemoglobin, and add a few drops of a
putrefied extract of meat. Close up hermetically, and allow to
stand some time at the ordinary temperature. The oxyhaamo-
globin will be reduced to haemoglobin, and if the solution is of
the proper concentration a crystallization of haemoglobin may
occur in the cylinder at lower temperatures. The solution
of haemoglobin is more purple than a solution of oxyhaemo-
globin, and shows a spectrum with only one absorption-band
occupying the space between the Fraunhofer lines D and E.
Carbon-monoxide Haemoglobin.
Preparation. — Pass a current of carbon monoxide (pre-
pared by heating a mixture of sulphuric and oxalic acids and
passing the gases through a wash-bottle containing KOH),
through a watery solution of oxyhaemoglobin. The color of
the solution becomes lighter, due to the formation of carbon-
monoxide haemoglobin. The spectrum of the solution is not
changed, but it differs from the oxyhaemoglobin in that it
cannot be reduced by reducing solutions into haemoglobin.
The crystals are isomorphous to the oxyhaemoglobin crystals,
but are less soluble.
22 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
MetliaemogloMn.
Preparation. — On treating a concentrated solution of oxy«
haemoglobin with a sufficient quantity of a concentrated
solution of potassium ferricyanide to give the mixture a
porter-brown color, methaemoglobin will be formed. After
cooling to 0° 0. add i vol. alcohol cooled to 0° C., and allow
the mixture to stand a few days in the cold. The crystals ob-
tained may be easily purified by recrystallization from water
by the addition of alcohol. (Pig's blood is better adapted for
the preparation of methsemoglobin than dog's blood.)
Properties.- — Methaemoglobin crystallizes in brownish-red
needles, prisms, or six-sided plates. It dissolves readily in
water, and this solution becomes red on the addition of alkali.
The spectrum of methaemoglobin shows three absorption-
bands, one in the red about half way between C and D, the
other two between D and E, which resemble the position of the
oxyhgemoglobin lines, but on careful measurement are found
to be different.
Fibrin Ferment.
Preparation. — Blood-serum is mixed with 10 to 15 times
its volume of absolute alcohol ; by this means the proteids are
precipitated, as also the ferment. The precipitate is allowed
to stand a few months with the alcohol, which renders the
proteids insoluble. Filter the precipitate off and dry in de-
siccator over sulphuric acid. The ferment is separated from
the other bodies by extracting with water, which dissolves the
ferment. (A. Schmidt.)
Properties. — A solution of the ferment will coagulate peri-
cardial and similar fluids or solutions of fibrinogen. It is
most active at about 40° 0. It is diminished in action at 0° C.,
and entirely destroyed on heating its solutions to 73°-75° C.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 23
Glycocholic Acid, Ca6H4,N06,
Preparation. — 1. The precipitate obtained by treating an
alcoholic solution of dried decolorized bile with ether is dis-
solved in water, and dilute sulphuric acid added until a per-
manent and dense turbidity is produced. Allow this liquid
to stand a few hours, when glycocholic acid will separate out
as fine silky needles. Collect on filter, wash with water,
press between folds of filter-paper, and then dissolve in alcohol,
using as little alcohol for solution as possible. Add many
times its volume of ether, and glycocholic acid will separate
out as long silky needles.
2. Decolorized extract of ox-bile is dissolved in water and
the solution treated with a solution of lead acetate. A pre-
cipitate of lead glycocholate is produced. Collect on a filter,
wash, drain, and mix with alcohol. To this add a solution of
sodium carbonate in excess and evaporate to dryness. This
converts the lead glycocholate into sodium glycocholate.
Remove the sodium glycocholate from the lead glycocholate
by extracting the residue with absolute alcohol. Distil off
the alcohol nearly to dryness, and evaporate to dryness in a
porcelain dish. Dissolve this residue in water, decolorize
this solution by animal charcoal, and the glycocholic acid is
precipitated from the solution by the addition of dilute sul-
phuric acid. The acid thus obtained is purified by crystalli-
zation from boiling water on cooling, or by the addition of
ether to its solution in absolute alcohol.
3. Fresh ox-bile is treated with a few drops HC1, which
causes the precipitation of the mucoid substances. Filter,
and to each 100 c.c. filtrate add 5 c.c. strong HC1. Place in
a stoppered cylinder and add 30 c.c. ether for every 100 c.c.
bile, shake well, and allow to stand in a cool place. After a
time the mixture is found to be converted into a mass of
crystals of glycocholic acid. Filter and wash with ice-water
until the washings are colorless ; they are then dissolved in
24 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
the smallest possible quantity of boiling water, which deposits,
on cooling, crystals in a colorless and pure state. (Hilfner.)
Properties. — Glycocholic acid crystallizes in fine, colorless
needles or prisms. It is soluble with difficulty in water
(about 300 parts cold and 120 parts boiling water). Readily
soluble in strong alcohol, but with greater difficulty in ether.
The solutions have a bitter taste, but at the same time a
sweetish taste. The salts of the alkalies and alkaline earths
are soluble in alcohol and water, while the glycocholates of the
heavy metals are either insoluble or only sparingly soluble in
water. It is a monobasic acid. The solution of the alkali-
salts in water is precipitated by lead acetate, copper, ferric
salts, and silver nitrate. On boiling with acids or alkalies
it splits into cholalic acid and glycocoll. Glycocholic acid
or its salts are dextro-rotatory. Specific rotation of the acid
dissolved in alcohol is (a) D = -|- 29°.
Taurocholic Acid, C96H46NSO,.
Preparation. — Agitate dog's bile with animal charcoal and
alcohol, allow to settle, decant through filter. The clear
filtrate is evaporated to dryness and dissolved in a little hot
absolute alcohol and precipitated by the addition of ether.
This crystalline precipitate of sodium taurocholate is filtered,
dissolved in water, and precipitated by lead acetate and am-
monia, filtered, washed well, and then boiled with absolute
alcohol, filtered while hot, and the filtrate decomposed with
H2S until no further precipitation of PbS. occurs. This is
filtered, the filtrate concentrated by evaporation at a gentle
heat and then precipitated by an excess of water-free ether.
The precipitate consists of taurocholic acid which after a
time becomes converted into needle-shaped crystals possess-
ing a silky lustre. If the precipitate refuses to crystallize,
after a time add a drop or two of alcohol. (Parlce.)
Properties. — Taurocholic acid crystallizes in needles which
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 25
rapidly deliquesce in the air. It is readily soluble in water
and alcohol, yielding a solution with a bitter-sweetish taste.
Its salts are, as a rule, readily soluble in water, and the solu-
tions of the alkali salts are not precipitated by copper sulphate,
silver nitrate, or sugar of lead, but are precipitated, on the
contrary, by basic lead acetate. The aqueous solution of the
alkali salts foam like soap. On boiling with acids and alkalies
it splits into cholalic acid and taurin. Both aqueous and alco-
holic solutions of taurocholic acid are dextro-rotatory. Spe-
cific rotation of its alcoholic solution is (a) D = -{- 25°.
Cholalic Acid, C34H4006.
Preparation. — 1. 500 c.c. bile are treated with 75 grms.
barium hydrate and the mixture boiled for 24 hours on the
sand-bath. The flask should be connected with a return con-
denser. Allow the liquid to cool, and filter. Add concen-
trated hydrochloric acid to the filtrate, which decomposes the
barium cholalate, depositing impure cholalic acid. These
crystals are separated from the mother-liquor and pressed, and
dissolved in a solution of caustic soda; the solution is then
mixed with 30 grms. animal charcoal and allowed to stand for
a few days. It is next filtered and the filtrate decomposed with
hydrochloric acid, filtered again and washed thoroughly and
the substance from the filter dissolved by the smallest possible
quantity of hot alcohol. The alcoholic solution is treated with
water until a slight turbidity appears. On the liquid being
cooled cholalic acid separates in the form of hard, transparent
tetrahedra or in clumps of radiating needles. For further
purification recrystallize from methyl alcohol. (Mylius.)
2. Mix ox-bile with one fifth of its weight of a 30$ caustic-
soda solution and boil for 24 hours on water-bath, having the
flask connected with a return condenser. Now saturate the
liquid with C02 and evaporate to dryness. Extract the
residue with strong alcohol, which dissolves the sodium salts
26 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
of cholalic acid as well as those of choleic and stearic acids.
Now dilute with water until it does not contain more than
20$ alcohol, and add a dilute solution of barium chloride as
long as a precipitate occurs. Filter, and add to the nitrate,
which should yield no further precipitate with Ba012, hydro-
chloric acid. Allow the precipitate of cholalic acid to stand
with the liquid and it will be found to become crystalline;
purify by recrystallization from ethyl alcohol and then from
methyl alcohol. (Mylius.)
Properties. — Cholalic acid crystallizes with 1 molecule of
water in rhombic plates or prisms, or in larger rhombic tetra-
hedra or octahedra with 1 mol. alcohol of crystallization.
They are quite insoluble in water (4000 parts cold and 750
parts boiling), rather soluble in alcohol, but soluble with
difficulty in ether. It is somewhat soluble in glycerin and
almond-oil. Its solutions have a bitter-sweetish taste. The
water-free acid melts at 195° C. In the free state, as well as
in combination the acid is dextro-rotatory. The specific
rotatory power of the sodium salt is (a) D = +31.4°. The
watery solution of the alkali-salts, when not too dilute, is
precipitated immediately or after some time by sugar of lead
or by barium chloride. The barium salt, (C24H3906)2Ba,
crystallizes in fine, silky needles, which are soluble in 30 parts
cold and 23 parts boiling water, but more soluble in alcohol.
Grlycocoll or Glycocine, C2H5NO,.
Preparation. — 1 part hippuric acid is boiled from 10 to 12
hours, in a flask connected with a return condenser, with 4
parts dilute sulphuric acid (1 to 6). After allowing to cool
pour the contents of the flask into a porcelain dish and allow
to stand for 24 hours. Filter off the benzoic acid and wash
with cold water; concentrate the filtrate by evaporation, and
shake with ether to remove any traces of benzoic acid.
Dilute this acid solution and just neutralize with barium
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 27
hydrate, which precipitates the sulphuric acid ; allow the
precipitate to settle, wash by decautation with boiling water,
and concentrate the solution by evaporation. If an excess of
Ba(OH)2 has been added, remove this by passing CO, through
the solution, boiling and then filtering, and allowing to stand
until crystals commence to form. The crystals are removed
from the mother-liquor and this concentrated further, and
the process repeated as long as crystals continue to separate
out. The glycocoll is purified by recrystallization from water.
Properties. — Glycocoll occurs as fine, hard, colorless,
rhombic crystals or four-sided prisms. The crystals have a
sweetish taste, are soluble in 4.3 parts cold water, very slightly
soluble in spirits of wine, and insoluble in cold absolute alco-
hol and ether. It combines with acids, bases, and neutral
salts. Glycocoll dissolves copper hydroxide in alkaline solu-
tion, but does not reduce at the boiling temperature, but de-
posits on cooling dark blue needles if the liquid is sufficiently
concentrated. Its solutions possess an acid reaction. When
heated to 232°-236° C., glycocoll becomes brown, evolves gas-
bubbles, and melts. A solution of glycocoll is colored red by
ferric chloride. Its combination with HC1 is soluble in water
and alcohol.
Taurin, C2H,NS03.
Preparation. — Mix ox-bile with strong hydrochloric acid so
as to precipitate the mucoid substances, filter, and boil the
filtrate for several hours. After allowing to cool and to de-
posit the dyslysin and choloidic acid concentrate the liquid
on the water-bath, filter so as to remove any common salt or
other substances which may have separated. The filtrate is
treated with strong alcohol, which precipitates the taurin with
some NaCl. This precipitate is washed with alcohol, dried,
and dissolved in the smallest quantity of boiling water possi-
ble. On cooling the taurin deposits in fine four-sided prisms
which may be further purified by recrystallization from water.
28 HANDBOOK FOB BIO-CHEMICAL LABORATORY.
Properties. — Taurin crystallizes in colorless, often large,
shining, 4-6-sided prisms, which dissolve in 15-16 parts water
at the ordinary temperature, but rather more readily in warm
water. It is insoluble in alcohol arid ether. Taurin is taste-
less and its solutions are neutral to test-paper. On boiling
with strong caustic alkalies it yields acetic acid and sul-
phurous acid but no alkali sulphides; but this decomposition
does not occur on boiling with HC1 or HM)3. Taurin com-
bines with metallic oxides such as mercuric oxide. Metallic
salts do not precipitate solutions of taurin.
BiliruMn, C,.H18N,0S or Ca,H3eN,0,.
Preparation. — 1. Finely-powdered gallstones (of oxen) are
first extracted with ether and then with boiling water to remove
cholesterin and bile-acids. The residue is treated with dilute
hydrochloric acid, which sets the pigment free. The insoluble
powder is thoroughly washed with water and alcohol and then
dried and boiled with chloroform until entirely extracted.
Evaporate the chloroform solution on the water-bath and treat
the powdered residue with absolute alcohol and ether, which
removes the Ulifuscin. Dissolve the remaining residue, insolu-
ble in alcohol, in chloroform; precipitate the bilirubin from
this solution by the addition of alcohol, and repeat this several
times. The bilirubin is finally dissolved in chloroform and
allowed to evaporate spontaneously, when crystals of bilirubin
separate.
2. Dilute bile (preferably from the dog) with water, and
precipitate with milk of lime. Pass carbon dioxide through
the mixture, filter and wash. Suspend the precipitate in water,
decompose it with hydrochloric acid and shake with chloro-
form, care being taken to avoid an excess of air. The chloroform
solution is evaporated to a very small volume, precipitated by
alcohol, and purified by repeated solution in chloroform and
reprecipitating with alcohol.
HANDBOOK FOB BIO-CHEMICAL LABORATORY. 29
Properties. — Bilirubin occurs partly amorphous and partly
crystalline, as rhombic plates whose obtuse angles are often
rounded. It is insoluble in water, almost insoluble in ether,
and very sparingly soluble in alcohol; easily soluble in chloro-
form, especially in the warmth, and less soluble in benzol, carbon
disulphide, amyl alcohol, fatty oils, and glycerin. Its solutions
show no absorption-bands and have, even on diluting greatly
(1 to 50,000), a decided yellow color. The combinations with
alkalies are insoluble in chloroform. Bilirubin forms com-
pounds with bases such as sodium, calcium, barium, lead, and
silver in ammoniacal solution. On allowing an alkaline solu-
tion of bilirubin to stand in contact with the air it gradually
absorbs oxygen, and green biliverdin is formed.
Biliyerdin, C16H18N,04 or C33HS6]S\08.
Preparation. — 1. Biliverdin is readily prepared by expos-
ing a thin layer of an alkaline solution of bilirubin to the air or
by passing a current of oxygen through the alkaline solution
until it has acquired a bright green color. Add hydrochloric
acid, which precipitates the biliverdin, filter, and wash the pre-
cipitate with water until no reaction for HC1 is obtained in the
filtrate. Dry the precipitate, dissolve in alcohol, and separate
the pigment by the addition of water to the alcoholic solution.
Any bilirubin present may be removed by extracting with
chloroform.
2. Add Pb02 to an alkaline solution of bilirubin and stir.
The liquid assumes a dark green color in a few minutes; now
acidify with acetic acid, which precipitates a compound of bili-
verdin with lead. This combination is decomposed by means
of alcohol containing sulphuric acid in solution. The alcoholic
solution containing the biliverdin is separated from the lead
sulphate by filtration and the filtrate poured into water, which
precipitates the biliverdin. Collect the precipitate on a filter
30 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
and purify by repeated solution in alcohol and reprecipitating
with water.
Properties. — Biliverdin is amorphous, but it .has occasionally
been obtained as green rhombic plates with tunicated ends. It
is insoluble in water, ether, pure chloroform, benzol, but
readily soluble in ethyl alcohol, methyl alcohol, glacial acetic
acid, and in chloroform containing alcohol. It dissolves in
alkalies, yielding a brownish-green solution which is precipi-
tated by acids, as well as by calcium, barium, and lead salts.
Solutions of biliverdin exhibit no definite absorption-bands
with the spectroscope. An absorption occurs from the red
toward the violet end of the spectrum.
Cholesterin, C26H440 + H20.
Preparation. — Powdered gallstones are first boiled with
water and then repeatedly boiled with alcohol. The solution is
filtered quickly through a heated filter, and the cholesterin
separates out in a fairly pure condition as the filtrates cool.
The crystals are washed with cold alcohol and then boiled
with an alcoholic solution of caustic soda, which saponifies
the fats present. After the evaporation of the alcohol, extract
the cholesterin from the residue by means of ether, which
dissolves the cholesterin, but not the soaps; filter and evap-
orate the ether, and purify the cholesterin by recrystallization
from a mixture of alcohol and ether.
Properties. — Cholesterin crystallizes from anhydrous ether
or chloroform as needles containing no water of crystallization,
but from alcohol as rhombic transparent plates whose sides
and angles often appear broken and whose acute angle is
76° 30' or 87° 30', with 1 mol. water of crystallization. It melts
at 145° C. when free from water of crystallization. Cholesterin
is insoluble in water, dilute acids, and alkalies. It is readily
soluble in boiling alcohol; also in ether, glycerin, chloroform,
and benzol, as well as the volatile and fatty oils. Its solutions
HANDBOOK FOE BIO-CHEMICAL LABORATORY. 31
are laevo-rotatory, the specific rotatory power being (a) D =
- 31.6°.
Ptyalin,
Preparation. — 1. Mixed human saliva is strongly acidulated
with phosphoric acid and the mixture carefully neutralized by
the careful addition of lime-water, which produces a copious
precipitate of calcium phosphate, and which carries down with
it a large proportion of the proteids present, together with all
the ptyaliu. On extracting the precipitate, after filtration,
with a Yolume of water equal to that of the saliva originally
employed, the enzyme passes into solution and is obtained
therefrom by the addition of absolute alcohol, which precipi-
tates it in the form of white flocculi, which when dried in
vacuo appears as a white powder. (Cohnheim.)
2. Saliva is diluted with an equal volume of water and
saturated with ammonium sulphate. The precipitate thus
formed is treated on the filter for 5 minutes with strong al-
cohol, removed from the filter, and further treated with ab-
solute alcohol for 1-2 days. It is now dried at 30° C. and
yields, on extraction with a volume of water equal to that of
the original saliva, a solution which is actively zymolytic and
free from proteids. (Krawkow.)
Pepsin.
Preparation. — 1. The mucous coat of the pig's stomach is
scraped with a watch-glass, and after carefully washing and
drying, by pressing between filter-paper, it is finely minced.
The mass is now digested with a 5$ solution of H3P04 at 35° C.
until nearly all is dissolved. Nearly neutralize the filtered
solution with lime-water, which causes a precipitate of calcium
phosphate, and which carries down with it the greater part of
the pepsin previously in solution. The peptones and para-
peptones remain in solution. The gelatinous precipitate is
carefully washed with water, pressed between filter-paper,
32 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
suspended in water, and treated with HOI until it just dis-
solves. The solution is then poured into a saturated solution
of cholesterin, made by dissolving it in a mixture of 4 parts
alcohol and 1 part ether. A precipitate of cholesterin will be
formed on the addition of the above acid solution, and this is
repeatedly shaken with the liquid, and the cholesterin will
mechanically carry down with it a part at least of the pepsin
in solution. Collect this on a filter, wash with water, then
with acetic acid, and lastly with water again until the wash-
water gives no further reaction for HOI with AgN03.
The moist cholesterin precipitate is shaken in a separatory
funnel with ether. Two layers will form — the upper an
ethereal solution of cholesterin and the lower an aqueous
solution of pepsin. Separate the two and shake the aqueous
solution repeatedly with ether until all traces of cholesterin
are removed. The turbid aqueous solution is now filtered,
and when acidulated possesses proteolytic activity. When
allowed to evaporate spontaneously it leaves a grayish, amor-
phous, non -hygroscopic nitrogenous body. (Brucke.)
2. The finely divided mucous membrane of the fundus of
the stomach is extracted with glycerin, or, better, with
glycerin containing 1 p.m. HC1. Add 10 to 20 parts glycerin
for every part by weight of the mucous coat and allow to
stand for 8 to 14 days. Filter and precipitate the pepsin from
the glycerin extract by the addition of a large excess of abso-
lute alcohol. This may be purified by dialysis through parch-
ment paper, (v. Wittich.)
Artificial Gastric Juice.
Preparation. — 1. The mucous membrane from the cardiac
region of five or six pigs' stomachs is finely divided and then
digested for two weeks, at 40° C., with two to three litres of
0. 5$ hydrochloric acid. At the end of this time all but traces
of albumoses have been converted into peptones, which are in
HANDBOOK FOE BIO-CHEMICAL LABORATORY. 33
solution together with pepsin, although a small portion of for-
eign matters, nucleins, antialbumid, etc., remain undissolved.
The liquid is filtered and saturated with powdered (KE4)2S04,
when a precipitation of the albumoses with the pepsin occurs.
The precipitate is collected on a filter, washed with a saturated
solution of ammonium sulphate, and then dissolved in 0.2$
HC1. Add 0.25$ thymol to this acid solution and dialyze in
running water until the whole of the (NH4)3S04 is removed.
On opening the dialyzing tubes a precipitate is found, which
is soluble in 0.2$ HC1 and furnishes a very active gastric
juice. (Eiihne and Chittenden.)
2. The mucous coat of a freshly removed pig^s stomach is
carefully washed with water and finely divided, or, if a calf's
stomach is employed, only the cardiac region is scraped with
a watch-glass or the back of a knife. The pieces of the
mucous coat or the slimy masses obtained by scraping are
rubbed up with pure quartz sand or glass powder, treated
with water, and allowed to stand for 24 hours in a cool place-
On filtering, an opalescent liquid is obtained, which, when
acidulated so as to contain 0.1-0.2 per cent HC1, possesses
powerful digestive activity. (Kuline.)
3. The stomach of a pig is opened, emptied, and then the
surface cleaned with a wet sponge (running water will dissolve
out a considerable part of the pepsin). The mucous mem-
brane is removed from all but the pyloric end of the organ.
It is then freed from a portion of the water adhering to it by
pressing between dry cloths and then mincing. The finely
divided mucous membrane is then placed in two or three litres
of dilute HC1 containing from 6 to 10 c.c. strong HC1 per litre,
and the mixture is digested in an incubator at a temperature
of 35°-45° C. for a period varying from a few hours to a day.
If sufficient fluid be present and the mixture now and then
shaken, all ought to be dissolved in a few hours, leaving but a
small quantity of brownish flakes and some mucus undis-
solved. The liquid is filtered through paper, and then may
34 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
be kept for several months without decomposition, retaining
active proteolytic properties. Such juice does not, however,
contain merely acid and pepsin, but considerable quantities
of albumoses and peptones. (Gamgee's Phys. Chem., vol. ii.,
page 82.)
Milk-curdling Ferment of the Stomach.
(RENNIN OR CHYMOSIN.)
Preparation. — Cut up five calves' stomachs finely, after
washing, and digest the mass for about 24 hours with a 0.5$
common-salt solution, keeping the temperature at about 30°
0., or less. After digestion filter, measure the filtrate, and
acidify with hydrochloric acid, so that it contains 0.1$ of the
acid. A thick precipitate of mucous matter is formed, and
to facilitate its easy separation the liquid is kept at 20°-30°
C. and allowed to stand until all of the mucous matter has
separated, or centrifugal force may be used. Filter, measure
the filtrate, and acidify with HC1 again so that the filtrate
contains 0.5$ acid, and now add powdered common salt until
no more salt dissolves. This supersaturated, acidulated salt
solution is brought to a temperature of 25°-30° C., and kept
at this temperature for 2-3 days, under constant agitation,
and then allowed to remain quiet for a day or so, the tem-
perature being raised to 30° to 35° C. A flocculent substance
is obtained, which rises to the surface of the liquid or is sus-
pended therein. This is carefully filtered off, and then dried
at a temperature of about 28° 0. The product thus obtained
is the pure zymotic product.'
Properties. — Rennin or chymosin is an amorphous, white,
gelatinous substance, greatly resembling aluminium hydrate.
It is without taste or smell, soluble in water, forming a limpid
or clear solution. Chymosin has the property of curdling
large amounts of milk at 37° C., or pure casein solutions
containing calcium salts,
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 35
Trypsin.
Preparation. — 1. The fresh pancreas ol an ox is freed from
adhering fat and connective tissue and then minced and di-
gested first with cold alcohol, and afterwards extracted with
boiling ether in an extraction apparatus. The insoluble resi-
due is then exposed to the air, so as to allow the ether to
evaporate, when there is left a white, friable mass. This
may be kept indefinitely, and made use of to prepare solu-
tions of trvpsin. (Called Killings pancreas powder.)
One part by weight of the above pancreas powder is digested
at 40° C. for 4 hours with 5 parts of 0.1$ salicylic acid solu-
tion. The residue after being squeezed is further digested for
12 hours with 5 parts of 0.25$ NaaC03 solution, and the resi-
due is again squeezed out. The acid and alkaline extracts
are mixed together, the whole made up to contain 0.25-0.5$
Ka2C03, and digested for at least a week in the presence of
0.5$ thymol. By this means all the first-formed albumoses are
fully converted into peptones ; this is essential. At the end
of the week the fluid is allowed to stand in the cold for 24
hours, filtered, faintly acidulated with acetic acid, and satu-
rated with neutral ammonium sulphate. By this means all the
trypsin is separated out and maybe collected on a filter, where
it is washed with (XH4)2S04 (saturated solution) till free
from peptones. It is now finally dissolved off the filter in a
little 0.25$ NAaC03 solution, to which thymol is added, and
thus an extremely active and pure digestive solution is ob-
tained. The ammonium sulphate present may be gotten rid
of by dialysis. (Kuhne.)
2. The pancreas is carefully removed from a dog killed 18
to 20 hours after a full meal and weighed. It is then pounded
up in a mortar with pure quartz sand or glass powder and
allowed to stand at the temperature of the laboratory for 24
hours and then mixed with 1 c.c. of 1$ acetic acid for every
gramme of pancreas, then for each part of the gland mass add
36 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
10 parts glycerin and allow to stand for three days. After
this time filter and precipitate the filtrate with alcohol and
wash with alcohol. On now dissolving the precipitate in
water we obtain a solution which has powerful digestive ac-
tion. (Heidenhain.)
3. A watery infusion of the gland may be made only after it
has been exposed to the air for 24 hours, adding 5 to 10 parts
water for each part by weight of the gland. Salkowski sug-
gests the digestion of the finely divided gland at 40° 0. with
water containing 5 to 10 c.c. chloroform per litre. After a few
days we obtain by this means an active solution of trypsin
which keeps.
Properties. — Trypsin is very soluble in water, but insoluble
in alcohol and glycerin. Its watery solution is not decom-
posed by long digestion at 40° C., and when evaporated it
yields a translucent, non-crystalline, yellowish solid residue.
Trypsin may be digested for a long time at 40° C. with caus-
tic soda without decomposing.
Diastatic Ferment of the Pancreas (Amylopsin).
Preparation. — 1. Finely divided pancreas, best after ex-
posure for 24 hours to the air, is dehydrated first by being
placed in strong alcohol and afterwards in absolute alcohol,
the action of which should be continued for some time. The
dry solid, separated from the alcohol, is then macerated in
glycerin. The glycerin solution is precipitated by the addi-
tion of alcohol, filtered, washed with alcohol, dried over sul-
phuric acid, and then dissolved in water, (v. Wittich.)
2. Precipitate the aqueous infusion of a pancreas which
has been treated with Mg2C03 with collodion, which carries
the proteids and the proteolytic ferment down with it in a
gelatinous form. The filtrate is concentrated by evapora-
tion in vacuo, and treated with strong alcohol, which throws
down a flocculent precipitate. Filter, and digest in a mixture
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 37
of equal parts alcohol and water, which dissolves the diastatic
ferment, a little tyrosin, and some salts, leaving some albumin
undissolved. The liquid is dialyzed, concentrated in vacuo,
and precipitated by absolute alcohol. The precipitate thus
obtained possesses feeble proteoly tic properties, due to remain-
ing traces of the proteolytic ferment, but has intense diastatic
properties. (Danielewslci.)
The Fat-decomposing Ferment of the Pancreas
(Steapsin).
Preparation. — A perfectly fresh pancreas is crushed in a
mortar with glass powder and mixed with a solution com-
posed of 90 c.c. glycerin and 1 c.c. of a \% solution of K"a2C03,
using 30 c.c. of the glycerin solution for every 3 grins, of the
pancreas. This is allowed to digest for not longer than four
to five days and then removed from the mass of the pancreas.
This extract has the property of decomposing neutral fats.
( Griitzner. )
Leucin, CaH13K02.
a. AMIDO-ISOBUTYL ACETIC ACID,
(CH3)2CH.CH2.CH(NHS).COOH.
Tyrosin, C9HnN03.
p. OXYPHENYL-AMIDO PfiOPIONIC ACID,
J00H
CHa.CH(NH2)COOH.
Preparation. — 1. These two bodies may be prepared in
large quantities by boiling albuminous bodies or albuminoids
with dilute mineral acids. Two parts hoof-shavings (J to 1
kilo in weight) are boiled for 24 hours with 5 parts concen-
trated sulphuric acid and 13 parts water, adding water from
time to time to replace that which has evaporated. After
38 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
boiling it is diluted with water and gradually treated with
milk of lime with constant stirring until the liquid is neutral
or a little alkaline. Filter through a folded filter, the residue
on filter boiled with water several times, and after filtration
added to the main filtrates. The several filtrates are concen-
trated by evaporation and oxalic acid added so as to precipi-
tate all the lime; filter, boil the calcium oxalate precipitate
with water several times, filter and unite all filtrates, evapo-
rate until crystallization begins, and allow to cool. What first
crystallizes out consists chiefly of tyrosin with only a little
leucin. Filter this off and concentrate the liquid more,
when a second crystallization will form on cooling, which
consists of leucin with some tyrosin. This operation of evap-
orating and crystallizing is continued until no more crystals
are obtained. The several batches of crystals are united and
boiled with a large quantity of water and enough ammonia
to dissolve them. To this hot solution add lead acetate, con-
stantly stirring, until the precipitate formed is no longer
brown, but white; filter, heat the yellow filtrate to boiling,
neutralize or make faintly acid with dilute sulphuric acid,
filter while boiling hot, and allow to cool. Pure tyrosin is
deposited while leucin remains in solution.
The mother liquor from the tyrosin is treated with H2S,
filtered and concentrated and boiled a couple of minutes with
freshly precipitated copper oxy hydrate. A deep blue solution
is the result, which, if filtered and concentrated, deposits sky-
blue crystalline warts, and an insoluble cuprous compound of
leucin. This precipitate, as well as the crystalline warts, is
decomposed in hot water with H2S, filtered, and the filtrate de-
colorized when necessary with animal charcoal, strongly concen-
trated, and allowed to crystallize. The leucin which deposits
may be purified by recrystallization from boiling alcohol or
by precipitating it as leucin lead oxide, decomposing this
precipitate when suspended in water with H2S, and evaporat-
ing the filtrate to point of crystallization. The tyrosin may
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 39
be purified by recrystallization from, boiling water or from
ammouiaciil water.
2. A large quantity of well-boiled fibrin is digested at
40° C. with a solution of Killings dried pancreas containing 1
per cent salicylic acid and some thymol. After a day or two
the liquid is filtered, faintly acidified with acetic acid, and
boiled. After filtration concentrate the filtrate nearly to
syrupy consistency and set aside to cool. Considerable quan-
tities of leuciu and tyrosin will have crystallized out after 24
hours. Remove the crystals and concentrate the mother-liquor
further if necessary, and precipitate the peptones with an
excess of hot alcohol, and filter while hot. On cooling crystals
of leucin will form if much is present. Pour oft the mother
liquor, and wash with a saturated solution of ammonium sul-
phate. The mixed leucin and tyrosin resulting from these
operations must be separated and purified by the method of
Hlasiwetz and Habermann, as described in method No. 1.
The leucin may be simplest separated from the tyrosin by
boiling the yellow crystalline masses or crusts with alcohol,
which dissolves the leucin and leaves the tyrosin in great part.
After filtering and concentrating the filtrate leucin crystal-
lizes out and is purified by repeated crystallization from
alcohol. Tyrosin is obtained in a crystalline form by dissolv-
ing the residue insoluble in alcohol with water containing
ammonia, and allowing this solution, after filtration, to evap-
orate at ordinary temperatures.
Properties. — Leucin crystallizes when pure in shining
white, very thin, doubly refractive plates, nodular masses or
balls, possessing a greasy feel and which float on water. Pure
leucin dissolves in 27 parts cold water, and in 1040 parts cold
and in 800 parts boiling alcohol, though the solubility seems
to vary with the source of the leucin. On slowly heating it
melts at 170° C., and sublimes in white, woolly flakes, which
are similar to sublimed zinc oxide. Its specific rotatory power
is (a) D = -j- 17.5°. Solutions of letiein in water are not, as a
40 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
rule, precipitated by metallic salts. The boiling hot solution
may, however, be precipitated by a boiling hot solution of
copper acetate. It forms crystalline compounds with sul-
phuric, hydrochloric, and nitric acids.
Pure tyrosin crystallizes in colorless, silky, fine needles,
which are often grouped in tufts or rosettes. From very
impure solutions it separates in part or wholly in nodules and
balls very like leucin. It is soluble with difficulty in water,
being dissolved by 2454 parts water at 20°C., and 154 parts
boiling water. It is insoluble in alcohol and ether. Tyrosin
is readily soluble in solutions of ammonia, caustic alkalies, and
alkaline carbonates. It is also soluble in dilute mineral acids,
with which it forms unstable compounds.
Aspartic Acid, C4H,N04.
AMIDO-SUCCIKIC ACID, C2H3(NH3)(CO.OH)2.
Preparation. — The mother-liquor from which leucin and
tyrosin have crystallized out (see page 38) is further concen-
trated and treated with a little alcohol, when, after some
time, new crystalline crusts will separate. These are dissolved
in water, and the solution boiled with freshly precipitated
cupric hydrate. On filtering the blue solution will deposit
the copper compound of aspartic acid, which occurs as light
blue needles. These are dissolved in HC1 anddecomposed by
H2S, when white crystalline plates of aspartic acid will sepa-
rate out. This is purified by crystallization from boiling water.
Properties. — Aspartic acid crystallizes in rhombic prisms
which are sparingly soluble in cold water or alcohol, but
readily soluble in boiling water. Its solution, strongly acid
with nitric acid, is dextro-rotatory, (a) D — -\- 25.16°, but in
alkaline solution it is laevo-rotatory. With copper it forms a
compound soluble in boiling water, but nearly insoluble in
cold water.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 41
Glutamic Acid, C5H9N04.
AMIDO-PYROTARTARIC ACID, C3H6(NH2)(COOH)2.
Preparation. — Boil 200 grms. casein (free from fat) with
600 c.c. concentrated LLCl and 500 grms. stannous chloride
for 3 days in a flask connected with a return condenser. After
diluting with 10 vols. water remove the tin by means of H2S,
and evaporate to syrupy consistency on water-bath. On stand-
ing in the cold the syrup will solidify into a mass of crystals;
these are stirred up and transferred to a filter connected with
a filter-pump, and as much mother-liquor sucked off as possi-
ble; then place the moist mass on an unglazed tile and placed
in desiccator until dry. Now recrystallize one or two times
from as little water as possible with the addition of cone.
HC1, and remove the mother-liquor as well as possible. The
crystals are now dissolved in a great deal of water and boiled
with an excess of lead oxyhydrate until the solution is free
from chlorine. Filter, precipitate the lead by H2S, filter again,
and evaporate the solution to point of crystallization. If these
crystals retain still small quantities of chlorine, treat the hot
solution with some silver oxide or carbonate; filter, treat the
filtrate with H2S, filter, and evaporate to crystallization.
Properties. — Glutamic acid crystallizes in the form of
small plates or rhombic tetrahedra or octahedra, which are
sparingly soluble in cold water, but readily in hot water. It
is insoluble in alcohol or ether., and melts at 135°-140°C. Its
dilute HC1 solution has a specific rotatory power of (a) D = -f-
31.1° to 31.6°. Dilute solutions reduce Fehling's solution.
When boiled with Cu(OH)2, glutamic acid separates in the
form of blue-colored prisms, which are soluble in 3400 parts
cold and -400 parts boiling water.
CH
Indol, C.H7K = C,H4 CH
H
\,
42 HANDBOOK FOR BIO-CHEMICAL LABOftATO&Y.
C.OH3
/ ^x
Skatol, 09H9N = C6H4 CH
X
or METHYL IKDOL.
Preparation. — Two kilogrammes of well-pressed blood fibrin
are placed in a large flask (12 litres capacity) treated with 8
litres water (containing 2 grms. KH2P04 and 1 grm. MgS04),
and well mixed with 200 c.c. of a cold saturated solution of
soda, then a few cubic centimetres of a putrefying infusion of
meat with some fragments of the decomposed meat. The flask
is closed with a stopper provided with a glass tube attached
with a rubber tube to a wash-bottle half full of water.
The rubber tube has a clamp which is left open during the
first days of the experiment. The mass is digested at 40° to
42° C. for 10 days, the flask being shaken from time to time
and the clamp closed as soon as the evolution of gas has
ceased and only opened now and then so as to liberate the
accumulated gases.
After this time the entire liquid contents of the flask is
distilled off until the residue in the flask measures 1 to 1.5
litres. The strongly ammoniacal distillate is acidified with
HOI and then precipitated by a solution of copper sulphate
and filtered. The clear filtrate is thoroughly shaken with
ether, which is best done by shaking fractions of -J litre at a
time in a separatory funnel drawing off the heavy liquid and
adding new portions of the filtrate to the ether in the funnel,
adding more ether from time to time. When one half of the
filtrate has been extracted with ether distill the ether from the
ethereal solution and use the ether for further extraction. The
entire ethereal extracts are distilled until about 500 c.c. are left.
This resi due is thoroughly shaken 2 times with a solution of
caustic soda to separate phenols and acids, and the ether now
distilled off at the lowest possible temperature. The oily
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 43
residue, previously treated with some caustic-soda solution, is
distilled in a current of steam* until no more indol passes over.
The distillate is now shaken with ether, the ethereal solu-
tion distilled at the lowest possible temperature, and the
residue allowed to evaporate in a deep vessel until, on being
allowed to cool, it solidifies as a crystalline mass. The latter
is then dried in a desiccator over sulphuric acid. (E. and H
Salkowski.) The crystals thus obtained, consisting of indol
and skatol, are dissolved in a very small quantity -of absolute
alcohol and then treated with 8-10 vols. water. The skatol is
precipitated, but not the indol. The skatol is filtered off and
recrystallized from ether. The indol is obtained from the
watery solution by extracting it with ether and allowing the
ether to evaporate spontaneously.
The mass of crystals, consisting of indol and skatol, may be
separated from each other by means of fractional distillation
in a current of steam when the skatol passes over first.
Skatol may also be obtained from this mixture of crystals
by dissolving about 0.5 grm. in a few drops of benzol and
adding 1.5 grm. crystallized picric acid and sufficient benzol
to dissolve all while being heated in a covered beaker-glass.
On cooling the mass solidifies to a red crystalline mass. Xow
add 2 vols. petroleum ether and stir, filter after 24 hours,
wash with petroleum ether, and allow the crystals to dry
in the air. Place the crystals in a distilling flask and
distill with dilute caustic soda, which decomposes the indol,
while the skatol passes over and is condensed. This skatol is
purified by recrystallization from ether.
Properties. — Indol crystallizes from hot aqueous solutions
* The current of steam is best produced by filling a large flask two-
thirds with water which has been acidified with a few cubic centimetres
of dilute H2SO4 and a few pieces of zinc, which by the action of the heat
develops a gentle current of hydrogen, which prevents bumping and
gives rise to a quiet, even generation of steam, which may be kept up
for hours.
44 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
as small scales, which melt at 52° C. It is tolerably soluble in
hot and less soluble in cold water ; readily soluble in alcohol,
ether, chloroform,, benzol, and petroleum ether. It forms a
combination with picric acid, consisting of red needles, which
are decomposed on heating with caustic soda, but pass over
without decomposition when distilled with ammonia. Indol
has a peculiar excrementitious odor.
Skatol crystallizes in small plates, which melt at 95° 0. It
is less soluble in water than indol, but in the presence of.
steam it distils readily. It is readily soluble in alcohol,
ether, chloroform, and benzol. With picric acid it forms a
crystalline compound, which does not decompose on being
heated with caustic soda, but passes unchanged into the distil-
late. It has an intense fetid odor.
Ordinary or Fermentation Lactic Acid,
C3HeOs or OH3.OH.OH.COOH.
Preparation. — 330 grms. cane-sugar and 0.5 grm. tartaric
acid are dissolved in 1750 c.c. boiling water and allowed to
stand for two days. Then add 4 grms. putrefying cheese
(German Handkase), 440 grms. sour milk, and 135 grms.
zinc white, and allow this mixture to stand for eight to ten
days at 40° 0. with constant shaking. After this time heat to
boiling, filter, and allow to cool, when zinc lactate will crystal-
lize out. This is purified by recrystallization and then de-
composed by H3S in hot aqueous solution, the solution fil-
tered, and the filtrate evaporated on the water-bath. The
syrupy residue is dissolved in ether (to separate any mannite),
and then the ether removed by distillation or by evaporation.
Properties. — Fermentation lactic acid has a similar appear-
ance to sarcolactic acid, namely, a colorless, faintly yellowish,
acid-reacting syrup, which mixes in all proportions with water,
alcohol, or ether. It is optically inactive. It forms salts
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 45
readily, which are soluble in water and most of them in alco-
hol. The zinc salt dissolves in 58 to 63 parts of water at
14-15° C. and contains 18.18$ water of crystallization, but is
insoluble in alcohol. The calcium salt dissolves in 9,5 parts
water and contains 29.22$ (= 5mol.) water of crystalliza-
tion,
Sarcolactic Acid,
PARALACTIC ACID, C3H603 orCH3.CH(OH).COOH,
Preparation* — Dissolve Liebig's extract of meat in 4 parts
of water and then add 8 parts 90$ alcohol, stirring all the
while, The mixture is allowed to stand and settle, and the
clear supernatant liquid is separated by decanfation. To re-
move any lactic acid from the insoluble residue, mix it with
twice its weight of warm water and precipitate by adding 4 or 5
times its volume of alcohol. The alcoholic solutions thus ob-
tained are evaporated on the water-bath to a thin syrup, and
this latter again precipitated by the addition of 3 or 4 times its
volume of alcohol. The alcoholic solution is now evaporated
to dryness, the residue mixed with water, some dilute H3S04
added, and then shaken several times with ether. On evapo-
rating the ethereal solution a residue is obtained which con-
sists of a mixture of sarcolactic and fermentation lactic
acids.
To separate the two above acids they are dissolved in water
and boiled with zinc carbonate or zinc white, the solution
filtered, and the filtrate evaporated until crystals commence to
form. The liquid is now treated with 4 or 5 times its volume
of 90$ alcohol ; after some time the liquid becomes turbid
and deposits needles, consisting of zinc sarcolactate, the zinc
salt of the other acid remaining in solution. The crystals are
collected on a filter, washed with absolute alcohol, dissolved
in water, decomposed by H2S, filtered, concentrated, shaken
with ether, and the pure acid obtained on the evaporation of
the ethereal solution.
46 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
Properties. — Sarcolactic acid occurs as a syrup, which dis-
solves in water, alcohol, and ether readily. It is dextro-rota-
tory, but its salts, on the other hand, are laevo-rotatory. Its
zinc salt crystallizes with 2 mol. (12.9$) water of crystalliza-
tion as small four-sided prisms, which dissolve in 17.5 parts
water, its calcium salt containing 24.83-26. 21$ (4 or 4.5 mol.)
water of crystallization, and dissolving in 12.4 parts water.
Protagon.
Preparation. — An ox-brain as fresh as possible, with the
blood and membranes carefully removed, is ground fine and
then extracted for several hours with 85$ alcohol at 45° C.,
filtered while hot, and the residue extracted with warm alco-
hol until the filtrate fails to yield a precipitate at 0° C. The
several alcoholic extracts are cooled to 0° C., and the precipi-
tates united and completely extracted with cold ether, which
removes cholesterin and lecithin-like bodies. The residue is
strongly pressed between filter-paper and allowed to dry over
sulphuric acid or phosphoric anhydride. The resulting mass
is powdered, moistened with water digested for many hours
with alcohol at 45° C., filtered, slowly cooled to 0° C. The
crystals may be purified by recrystallization. (Gamgee and
Blankenlwrn.)
Properties. — Protagon appears when dry as a white, loose
crystalline powder. It is soluble with difficulty in cold, but
more easily soluble in warm, alcohol and ether. At tempera-
tures above 55° C. its solutions decompose. It swells up in
little water, decomposes partly, and gives myaline forms. It
is soluble in glacial acetic acid, which deposits crystalline
forms on cooling. When boiled with a solution of Ba(OH)2,
protagon decomposes into glycero-phosphoric acid, fatty acids,
and neurine. It melts at 200° C., forming a brown syrup.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 47
Urea, CH4N20 or HaN.CO.NH2.
Preparation. — Evaporate ^ to 1 litre of urine to syrupy
consistency on the water-bath, allow to cool in ice-water, treat
while stirring with 3 vols. nitric acid of sp. gr. 1.3 which
has previously been boiled and then coolod to 0° C. Allow
this mixture to stand for a few hours at 0° C. The crystals
of urea nitrate which form are transferred to a filter of glass
wool and washed several times with small amounts of ice-cold
pure concentrated nitric acid, then dissolved in as small a
quantity of hot water as possible, and reprecipitated by cone.
HN03. These crystals are first drained, then dissolved in hot
water, and the solution decolorized by a little chlorine water,
and then treated with a small quantity of pure barium carbon-
ate until no more dissolves and effervescence ceases and the
solution is neutral. Now evaporate the solution to complete
dry ness on the water-bath, pulverize the residue, and extract
it with cold absolute alcohol, which dissolves the urea, but not
the barium nitrate. The filtered alcoholic solution is decolor-
ized when necessary by animal charcoal and evaporated at a
medium heat to point of crystallization.
Synthetical. — Heat 250 grms. coarsely powdered potassium
ferrocyanide in a capacious porcelain dish over a naked flame
with constant stirring until all is changed into a white pow-
der, leaving no lumps having a yellow nucleus. Care must
be taken not to apply too strong a heat, as otherwise the pow-
der will turn brown. The mass thus obtained is finely
powdered and thoroughly mixed with one half (-J) its weight
of dried finely powdered manganese dioxide, and the mixture
heated on an iron dish under the draught, constantly stirring
until deflagration begins and the mass becomes doughy. The
heat is continued until a portion of the mass placed in water
and acidified with HC1 fails to give a blue precipitate with
ferric chloride. Then allow to cool, lixiviate with cold water,
add to the solution f of the weight of the drie_d potassium
t
48 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
ferrocyanide of ammonium sulphate, filter, and evaporate at
about 60°-70° 0., at which temperature the ammonium cya-
nate is converted into urea. The crystals of potassium sul-
phate are removed from time to time and then the solution
evaporated to dryness. The urea is obtained from this residue
by extraction by absolute alcohol as above described.
Properties. — Urea crystallizes in needles or in long, colorless
four-sided, often hollow, anhydrous rhombic prisms, similar
to saltpetre. It has a neutral reaction, has a bitter taste, and
produces a cooling sensation on the tongue. It melts at 130°-
132° 0., but partly decomposes at about 100° C. At ordinary
temperatures it dissolves in equal weights of water and in five
parts alcohol. Urea is readily soluble in amyl alcohol. It is
insoluble in anhydrous ether, chloroform, and in petroleum
ether. Urea combines with salts, such as NaCl, NH4C1, and
the chlorides of the heavy metals, such as mercury, gold, zinc,
copper, etc., producing crystalline combinations. It also com-
bines with acids, both inorganic and organic, forming crystal-
line salts.
Uric Acid,
C6H4N403 or HN— CO
-NH
AoL:
I
HN— C— NH
Preparation. — 1. Powdered Peruvian guano is just heated
to boiling with 15-20 parts water, in a porcelain dish, and
then dissolved by the careful addition of a small amount of
concentrated solution of caustic soda or potash, and now
boiled until the odor of ammonia has disappeared. Filter and
saturate the filtrate with carbon dioxide, when a gelatinous,
afterwards granular, precipitate of acid potassium urate is
HANDBOOK FOR BIO-CHEMICA
formed. The liquid is decanted after standing 24 hours, and
the precipitate washed with small quantities of ice-cold water,
and while still moist added to boiling dilute hydrochloric acid
and kept boiling for some time, continually stirring. After
cooling the separated uric acid is filtered, thoroughly washed
with cold water, and dried. If not quite pure, it can be redis-
solved in alkali, precipitated by C02, and treated again as
above, or it may be added to water and dissolved in the cold
by the gradual addition of sodium amalgam, and after the re-
moval of the impurities by filtration the uric acid may be re-
precipitated by HOI.
2. Filtered normal urine is treated with 20-30 c.c. of 25$
HC1 for each litre of urine. After 48 hours collect the crys-
tals and purify them by redissolving in dilute alkali, decolor-
izing with animal charcoal, and reprecipitating with hydro-
chloric acid. Filter, wash with ice-cold water, and dry.
3. Uric acid may be prepared synthetically by melting
0.1-0.15 grms. glycocoll with 1-2 grms. urea in a test-tube
over a small flame. The mass is heated until solid, care be-
ing taken not to apply too strong (above 220° C.) a heat, also
not too low a heat. The cooled mass, which ought to be yel-
low or yellowish-brown, is dissolved in boiling water with the
addition of ammonia and filtered; the filtrate is precipitated
when cold with a mixture of magnesium mixture and ammo-
niacal silver nitrate solution. The precipitate is washed with
ammoniacal water, subdivided in hot water, and treated with
a solution of sodium sulphide. The filtrate from the silver
sulphide is acidified with HC1 and concentrated, when on
cooling impure uric acid will crystallize out. Test these crys-
tals with the murexid test, and if they do not give the reac-
tion reprecipitate with ammoniacal silver solution and proceed
as above, when comparatively pure uric acid will be obtained.
It is better to make several fusions of glycocoll with urea, and
adding these together and obtaining the uric acid therefrom
in larger quantities. (/. Horbaczeivski.)
50 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
Properties. — Pure uric acid is a white, odorless, and taste-
less powder consisting of very small rhombical prisms or
plates. It is insoluble in alcohol and ether, rather easily sol-
uble in boiling glycerin, very difficultly soluble in cold water
(14000-15000 parts), and difficultly soluble in boiling water
(1800-1900 parts). It is soluble in sulphuric acid in the cold
without decomposition, and readily soluble in many salts of
the alkalies, as in the caustic alkalies; ammonia, however,
scarcely dissolves it. Uric acid reduces alkaline solutions of
copper or silver, but not alkaline solutions of bismuth.
Hippuric Acid,
C9H9NO, or C6H5.CO.NH.CHa.COOH.
Preparation. — 1. Treat fresh horse or cow urine with milk
of lime until it is of a strong alkaline reaction; warm, filter,
evaporate the filtrate to a syrupy consistency, and acidify
strongly with HC1 when cold. The hippuric acid thus pre-
cipitated is drained, washed with cold water, pressed between
filter-paper, dissolved in as small a quantity of boiling water
as possible, filtered, and the boiling-hot filtrate treated with
chlorine gas until the color of the solution is pale yellow.
Cool quickly, filter, wash the hippuric acid a few times with
cold water, and crystallize from boiling water after treating
the solution with animal charcoal. (Tfi. Curtius.)
2. One litre of the fresh horse or cow urine is made faintly
alkaline with sodium carbonate and filtered, the filtrate
evaporated nearly to dryness, and the residue repeatedly ex-
tracted with cold, strong alcohol. The alcohol is nearly en-
tirely distilled off from the extracts the remaining watery
solution acidified with HC1, and then repeatedly extracted
with fresh portions of acetic ether (at least five times). The
acetic ether extracts are united, and washed by shaking with
water in a separatory funnel, and then evaporated at a moderate
temperature. The residue is repeatedly treated with petro-
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 51
leum ether, which removes the benzole acid, oxyacids, fat, and
phenol, while the hippuric acid is left undissolved. The
residue is dissolved in a little warm water, treated with
animal charcoal, and evaporated at 50°-60° C. to point of
crystallization. (Bunge and Schmiedeberg.)
Synthetical — Hippuric acid may be prepared by adding a
few drops caustic-soda solution to a watery solution of glyco-
coll, shaking with benzoyl chloride, adding gradually an ex-
cess thereof and then making strongly alkaline with caustic
soda. Now precipitate with hydrochloric acid on cooling.
The precipitate of hippuric and benzoic acids are carefully
drained, pressed between filter-paper and washed with cold
water, dried, and extracted with petroleum ether, which only
dissolves the benzoic acid. The remaining hippuric acid is
recrystallized from hot water. (J. Baum.)
Properties. — Hippuric acid crystallizes in semi-transparent,
milk-white, long, four-sided rhombic prisms or columns, or
in needles on rapid crystallization. They dissolve in 600
parts cold water, but more easily in hot water. It is soluble
in alcohol, but with difficulty in ether. It is insoluble in
petroleum ether, benzol, and carbon disulphide. Hippuric
acid is readily soluble (about 12 times) in acetic ether. It
combines with bases, forming salts which are soluble in water
or alcohol. The combinations with the alkalies and the alka-
line earths are readily soluble in alcohol, while the silver,
copper, and lead salts are soluble with difficulty. On boiling
with caustic alkalies, mineral acids, or by continued heating
with water at 170°-180° C. it splits into benzoic acid and
glycocoll.
Allantoin,
.CH.NH.CO.NH,.
Preparation. — 1. Evaporate the amniotic fluid of a cow to
one sixth (-J-) its volume, or calf urine to syrupy consistency,
52 HANDBOOK FOE BIO-CHEMICAL LABORATORY.
and allow it to stand in a cold place for several days, when
allantoin and magnesium phosphate crystallize out, with ge-
latinous magnesium urate. Now dilute with water, stir, and
decant the liquid from the allantoin and magnesium-phos-
phate crystals. Eepeat this several times, and boil the crystals
with water after having added some animal charcoal; filter
while hot; faintly acidify the filtrate with HC1, which keeps
the phosphates in solution, and allow to crystallize. Purify
by recrystallization from water. ( W'ohler.)
2. Make a thin paste of uric acid and water, heat nearly to
boiling, and add in small quantities a thin paste of finely
ground lead peroxide with water, continually stirring until
the chocolate-brown color of the mixture just disappears.
(Care must be taken not to have an excess of lead peroxide.)
If the mass has become too thick, some water may be added
during the operation; now filter while hot, and allow to cool.
After crystallization the mother-liquor is concentrated by
evaporation so as to obtain more allantoin. The preparation
is purified by repeated crystallization from small amounts of
boiling water. (K. Hoffmann.)
3. Treat 3 mols. uric acid, subdivided in water, with 1 mol.
potassium permanganate, taking care that the mixture does
not get warm. Filter quickly, supersaturate with acetic acid
and allow to stand 24 hours, when crystals of allantoin will be
formed. (K. Hoffmann?)
Properties. — Allantoin is a colorless substance, often crys-
tallizing in prisms, or, when prepared from calf urine, as small,
thin columns. It is soluble in 160 parts cold (20° C.) and 30
parts boiling water. It is insoluble in cold absolute alcohol
and ether. Allantoin combines with acids and bases, forming
salts. "With silver oxide and mercuric oxide it forms im-
portant combinations. Allantoin reduces Fehling's solution
on continued boiling.
HANDBOOK FOE BIO-CHEMICAL LABOBATOBY. 53
Creatin,
C4H9N3Oa + H20 or NH. : C [NHJ.N[CH3].CH2.COOH.
Syn. METHYL GUAKIDIH ACETIC ACID.
Preparation. — 1. Beef extract (Liebig's) is treated with
water and the albumin removed as far as possible by heating
to boiling, and filtering. The filtrate is precipitated by the
careful addition of basic lead acetate, taking care not to add
too great an excess of the precipitant. Filter, remove the
excess of lead acetate in the filtrate by HaS, and evaporate the
nitrate to a small volume at moderate temperature. Allow
the solution to stand one week in a cool place, filter off the
separated crystals, wash with 88$ alcohol, and purify when
necessary by recrystallization. The crystals may be decoloF-
ized by dissolving them in hot water to which animal charcoal
has been added, filtering,and allowing the filtrate to crystallize.
2. Finely chopped meat is mixed with one half its weight
of water, pressed, and the residue again extracted with the
same amount of water. This extract is freed from albumin
by heating to boiling and filtering, and adding a solution of
baryta water to the filtrate as long as a precipitate forms.
Filter off the precipitated phosphates, remove the excess of
baryta by means of carbon dioxide, filter, and evaporate the
filtrate to one twentieth of its volume on the water-bath. The
syrup is allowed to stand several days in a cool place when
crystals will form. If a skin should form on the surface this
should be removed.
3. Chopped meat is treated with ether and allowed to stand
for a few days, when a strongly acid fluid separates out. This
is colored red, due to myoheematin. This watery fluid is sep-
arated and evaporated, when crystals of creatin will separate
out, which may be purified by recrystallization as above.
(MacMunn.)
Properties. — Creatin crystallizes in hard, colorless, shining,
54 HANDBOOK FOE BIO-CHEMICAL LABORATORY.
monoclinic prisms containing 1 mol. water of crystallization,
which it loses when heated to 100° C. It dissolves in 74
parts water at the ordinary temperature, and in 9400 parts
absolute alcohol. It is insoluble in ether. Its watery solu-
tion is neutral in reaction, and has a bitter taste. Creatin
forms crystalline compounds with mineral acids and with
mercury. When heated with dilute mineral acids it is con-
verted into creatinin. It forms compounds with certain
metallic solutions.
Creatinin,
METHYL GUANLDHST HYDANTOIK.
Preparation. — 1. Evaporate several litres of urine to one
third or one quarter its volume, allow to cool, and decant liquid
from the precipitated salts, now precipitate with lead acetate
and filter. The excess of lead in the filtrate is removed by H2S
or soda solution; filter again, nearly neutralize with soda solu-
tion or acetic acid, heat to drive off H2S, and add a concentrat-
ed solution of mercuric chloride, which causes a precipitate.
Filter, suspend the precipitate in water and decompose it
with a current of H2S, and again filter. Decolorize the filtrate
with animal charcoal and evaporate to dryness. The mass is
recrystallized several times from strong alcohol. The creatinin
may be easily obtained pure from the creatinin hydrochloride
by treating with lead oxyhydrate and then crystallizing.
2. One-half to one litre of urine is treated with baryta mix-
ture (1 vol. saturated Ba(N03)2 solution and 2 vols. satu-
rated baryta-water) until no further precipitate is formed,
filtered, and the filtrate evaporated on the water-bath to a
thin syrup. This is mixed with an equal volume alcohol and
allowed to stand in a cool place for 24 hours ; the salts which
separate out are removed by filtration and the filtrate treated
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 55
with 1 to 2 c.c. concentrated alcoholic solution of acid-free
zinc chloride. After a few days yellow crystals of creatinin
zinc chloride separate out ; these are filtered off, washed with
alcohol, dissolved in hot water and decomposed by boiling for
about half an hour with an excess of freshly precipitated
lead oxide or lead carbonate. Filter while hot, decolorize the
filtrate by boiling with animal charcoal, filter again, evaporate
to dryness, and extract the residue by strong alcohol in the
cold, which removes the creatinin. Creatin remains behind
undissolved.
3. Precipitate -J to 1 litre of urine with phospho-tungstic
acid and hydrochloric acid, filter, and treat the precipitate
with caustic baryta. Filter, and remove the excess of baryta
by means of carbon dioxide, and filter again. Evaporate the
filtrate to dryness, extract the residue with strong alcohol and
evaporate the alcohol, when the creatinin will be obtained
impure. It may be purified as above described.
Properties. — Creatinin crystallizes in long, colorless, highly
refractive, rnonoclinic prisms, which do not become white
with loss of water when heated to 100° C. It dissolves in
11.5 parts cold water and 100 parts cold alcohol, though it is
more soluble in hot liquids. It is nearly insoluble in ether.
Creatinin combines with HC1 and certain metallic salts, form-
ing crystalline compounds. Creatinin acts as a reducing
agent, reducing Fehling's and Trommer's solutions; also
reducing mercuric oxide to metallic mercury, and yielding
oxalic acid and methyl guanidin.
X a n thin Bases.
HYPOXAXTHIX, C5H4N40.
ADEXIX, C5H6N6.
GUANIN, C&H5N50.
Preparation, — 250 grms. finely chopped beef liver is placed
56 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
in a large flask, provided with a glass stopper, and treated
with 2.5 litres chloroform water (2.5 litres tap water with
12.5 c.c. chloroform, and shaken until the latter is dissolved);
add 2.5 c.c. chloroform, and place this mixture in an incu-
bator and allow to digest at 40° C. for two to three days, con-
tinually shaking. After digestion transfer to a large dish
and heat to boiling, and continue boiling after acidifying with
acetic acid until the albumin is precipitated. Now filter, and
evaporate the filtrate to 800-1000 c.c. Make alkaline with
ammonia, filter off the precipitate formed, and completely
precipitate with a 3$ silver-nitrate solution. The gelatinous
precipitate formed consists of the silver combination of the
xanthin bases. Care must be taken not to have a silver-
chloride precipitate form, otherwise more ammonia must be
added. Filter off the precipitate, wash well, place the pre-
cipitate in a flask while still moist, and dissolve by the aid of
heat in nitric acid of sp. gr. 1.1 (equal volumes nitric acid
and water), adding a little urea to prevent the formation of
nitrous acid. The solution should be nearly clear. Filter
while hot and allow to stand for 24 hours. A precipitate of
guanin, adenin, and hypoxanthin silver nitrate will have
formed, while xanthin silver nitrate remains in solution.
Filter and wash with dilute nitric acid.
Add ammonia to the filtrate until alkaline, when a precipi-
tate of xanthin silver nitrate will form as a brownish or red-
dish flocculent precipitate. Filter, wash, suspend in water
with a few drops ammonia; heat, add a few drops ammonium
sulphide, stir, filter off the silver sulphide, and evaporate the
filtrate, when xanthin will be obtained. (The silver com-
pound may also be decomposed by HC1, but then on evapo-
ration xanthin hydrochloride is produced.) The xanthin
obtained as above is never quite pure.
The residue of hypoxanthin, guanin, and adenin silver
compounds is suspended in water and decomposed with a
current of HaS, filtered, and the filtrate evaporated to a
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 57
small volume. This is made alkaline with ammonia and
allowed to stand, when a deposit of guanin will occur, while
the hypoxanthin and adenin (besides ammonium nitrate)
remain in solution. Filter off the guanin and wash with
water. The filtrate is evaporated to dryness on the water-
bath and the residue extracted with small amounts of water
to remove the ammonium nitrate, leaving the insoluble hypo-
xanthin and adenin.
Properties. — Xanthin is amorphous, or forms masses of
crystalline leaves. It is soluble with difficulty in water (in
14,151 to 14,600 parts water at -f 16° C. and in 1300 to
1500 parts at 100° C.). It is insoluble in alcohol or ether,
but is dissolved by alkalies or acids. Xanthin forms a crys-
talline, difficultly soluble combination with HC1. With very
little caustic soda, xanthin gives a crystalline combination
which is readily soluble in more alkali. When dissolved in
ammonia, xanthin gives a gelatinous precipitate of xanthin
silver, with silver nitrate. This body is soluble in nitric acid.
A watery solution of xanthin is precipitated on boiling with
copper acetate. Xanthin is precipitated at the ordinary tem-
peratures by mercuric chloride and also by ammoniacal basic
lead acetate.
Hypoxanthin occurs as colorless, crystalline needles, which
are soluble in 300 parts cold and 78 parts boiling water. It
is nearly insoluble in alcohol, but is soluble in acids or alkalies.
The combination with HC1 is crystalline, but is more soluble
than the corresponding combination with xanthin. The
silver combination with hypoxanthin dissolves with great
difficulty in boiling nitric acid. Hypoxanthin picrate is diffi-
cultly soluble, but if a boiling solution is treated with a neu-
tral or faintly acid solution of silver nitrate, the hypo-
xanthin is quantitatively precipitated as a combination, hav-
ing the formula CbH3AgX4O.C6Ha(XOa)3OH.
Adenin crystallizes with 3 rnol. water of crystallization in
long needles, which become opalescent when heated or ex-
58 HANDBOOK FOR BlO-CHEMtCAL LABORATORY.
posed to the air. When the crystals are slowly heated with
insufficient water from a solution they become suddenly opa-
lescent at -j- 53° C. It is soluble in 1086 parts cold water,
and much more readily soluble in hot water. Adenin is
insoluble in ether, but somewhat soluble in hot alcohol. It is
readily soluble in acids or alkalies. It is more soluble in
"ammonia than guanin, but less soluble than hypoxanthin.
The silver combination of adenin is difficultly soluble in
warm nitric acid, and deposits crystals of adenin silver nitrate
on cooling. It also gives a combination with picric acid
which is soluble with difficulty.
Guaniu forms an amorphous, colorless powder, but crystal-
lizes in very small crystals from a solution in concentrated
ammonia if allowed to spontaneously evaporate. It is insolu-
ble in water, alcohol, or ether, readily soluble in mineral
acids or alkalies, but with difficulty in ammonia. The HC1
salt of guanin crystallizes readily, and on account of its action
on polarized light may be made use of in the identification
of guanin. Picric or metaphosphoric acids precipitate even
very dilute solutions of guanin. The silver combination is
very difficultly soluble in boiling nitric acid, and deposits
crystals on cooling.
UroMlin.
Preparation. — 1. Several litres of urine are acidified with
H2S04, using 2 grms. for every litre; then saturated with
ammonium sulphate, filtered, and washed with a saturated so-
lution of ammonium sulphate faintly acidified with sulphuric
acid. The residue of urobilin is dissolved in alcohol, which
requires some time. The filtered alcoholic solution is mixed
with chloroform and a volume of water equal to the alcoholic
solution, well shaken together, and allowed to stand until the
chloroform has settled. The chloroform is removed by means
of a separatory funnel, and washed with twice its volume of
water. The clear chloroform solution is now filtered through
HANDBOOK FOR BIO-CSEMICAL LABORATORY. 59
a dry filter into a distilling flask and the chloroform distilled
off. The residue in the flask is washed with ether, which
only dissolves very little of the urobilin. This residue is
again dissolved in chloroform, filtered, and allowed to evapo-
rate at a very moderate temperature. (Melm.)
2. Precipitate the urine with basic lead acetate, wash the
precipitate with water, dry at the ordinary temperature, and
then boil it with alcohol, acidified with sulphuric acid. The
filtered alcoholic solution is diluted with water, now satu-
rated with ammonia, and then treated with a zinc-chloride
solution. This new precipitate is washed free from chlorine
with water, boiled with alcohol, dried, dissolved in ammonia,
and this solution precipitated with sugar of lead. This pre-
cipitate, which is washed with water and boiled with alcohol, is
decomposed by alcohol containing sulphuric acid; the filtered
alcoholic solution is mixed with -J vol. chloroform, diluted
with water and shaken repeatedly, but not too energetically.
The urobilin is taken up by the chloroform. This last is washed
once or twice with a little water and then filtered, leaving the
urobilin on evaporation of the chloroform. It may be further
purified by treating with ether.
3. The urine is treated with ammonia in not too great ex-
cess, filtered, and the filtrate precipitated with a concentrated
watery or alcoholic solution of zinc chloride. If the filtrate
from this precipitate is still deeply colored, precipitate
by adding more ammonia and filtering. The voluminous,
generally red or reddish-brown precipitate is washed free
from chlorine by first using cold and then hot water, now
boiling with alcohol, and drying at a low temperature. The
mass is powdered and dissolved in ammonia, leaving a small
residue, and this ammoniacal solution is precipitated with
sugar of lead, and the red precipitate filtered off and washed
with water until the coloring matter commences to go into
solution. The precipitate is now digested with alcohol acidi-
fied with sulphuric acid, filtered, and the filtrate repeatedly
60 HANDBOOK FOE BIO-CHEMICAL LABORATORY.
shaken with its J vol. chloroform and considerable water. The
chloroform is separated and washed once or twice with water
and the chloroform now distilled off. The residue, consisting
of impure urobilin, is washed with ether, which removes
considerable quantities of a red coloring matter, and leaves
the urobilin as brown amorphous masses.
Properties. — Urobilin, according to Jaffe, is amorphous, red,
dingy red or reddish yellow, according to the method of prep-
aration. It is readily soluble in alcohol, amyl alcohol, acetic
ether, and chloroform, but less readily in ether or water. It
is soluble in alkalies, and is incompletely precipitated from
the alkaline solution by the addition of acid. Its alkaline
solutions give insoluble combinations with salts of the heavy
metals, such as zinc and lead. A neutral solution of urobilin
gives a green fluorescence, and the acid solution shows a faint
absorption-band between b and F, which borders on F, or in
greater concentration extends over F. The alkaline solutions
show a darker or more sharply defined absorption-band, almost
midway between b and F.
Glycuronic Acid,
C6H1007.
Preparation. — Puree or Indian yellow is rubbed fine with
water in a mortar, forming a thick mass. This is acidified
with hydrochloric acid, filtered, and thoroughly washed with
water. The euxanthic acid thus obtained is dissolved in hot
alcohol, filtered, and allowed to stand to crystallize as beautiful
yellow needles. This crystallization is repeated again so as to
obtain pure euxanthic acid. One part of the euxanthic acid
is treated with 150 parts water and heated, in a Papin's di-
gester supplied with a thermometer, first to boiling, allowing
the cover of the digester to be open. After closing the cover
heat for an hour, keeping the temperature at 120°-125° C.
On cooling the liquid is filtered, leaving the crystals of unde-
HANDBOOK FOR BIO-CHEMICAL LABORATCEY. 61
composed enxanthic acid and euxanthon on the filter. These
are again treated with water as above. The filtrate is evapo-
rated in a flat dish, care being taken not to have too high a
temperature. When at a syrupy consistency it is allowed to
stand, when crystals of glycuronic acid anhydride are obtained.
(Thierf elder.)
Properties. — Glycuronic acid is not crystalline, but is ob-
tained only as a syrup. It dissolves in alcohol, and is easily
soluble in water. It forms crystalline salts with potassium
and sodium. The neutral lead salt is soluble in water, but
the basic salt is, on the contrary, insoluble. The acid is dex-
tro-rotatory (a)D = -f 19.4, and reduces alkaline solutions of
copper, silver, and bismuth salts. It gives a crystalline com-
bination with phenyl hydrazine, melting at 114°-115° C.
LIST OF
IMPOETANT TESTS
ARRANGED IN ALPHABETICAL ORDER.
LIST OF IMPORTANT TESTS ARRANGED IN
ALPHABETICAL ORDER.
ACETONE. See Chautard, Gunning, Lieben, Legal, Le
Nobel, Malerba, Penzoldt, Reynolds.
ACETO-ACETIC ACID. See Gerhardt.
ADAMKIEWICZ'S Keactiou (Proteids). — Add the proteid to
a mixture of 1 vol. concentrated sulphuric acid and 2 vols.
glacial acetic acid. A reddish-violet color is obtained slowly
at the ordinary temperature, but more quickly on heating.
The liquid has also a feeble fluorescence, and gives an ab-
sorption band between the lines b and F in the solar spec-
trum.
ALBUMIN. See Axenfeld, Boedeker, Cohen, Fnrbringer,
Heller, Heijnsius, Hindenlang, Johnson, McWilliam, Mehu,
Oliver, Pavy, Reoch, Roberts, Rees, Raabe, Spiegler, Tanret,
Zouchlos.
ALLEN'S Test (Phenol). — Add to one to two drops of the
liquid to be tested a few drops of hydrochloric acid and then
1 drop of nitric acid. Cherry-red color is produced, which
is intensified by gentle warming. Alcohol does not interfere
with the reaction. On supersaturating with caustic soda the
red liquid becomes dark brown,
66 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
ALMEN'S Reaction (Blood). — Mix in a test-tube equal vol-
umes of tincture of guaiacum and old turpentine which lias
become strongly ozonized by the action of air under the in-
fluence of light. Allow the liquid to be tested to flow down
gently on the surface of this mixture. If blood or blood -
coloring matters are present, a bluish-green and then a beau-
tiful blue ring appears where the two liquids come together,
and if shaken the liquid becomes more or less blue. Pus
gives a blue color with this mixture, but in this case the tinct-
ure of guaiacum alone, without turpentine, is colored blue.
ALMEN'S Test (Glucose). — Heat liquid with a solution of
bismuth subnitrate dissolved in caustic soda and Rochelle
salts. If glucose is present, the liquid becomes dark,
cloudy, dark brown, or nearly black, and non-transparent.
After a time a black deposit appears.
ANDREASCH'S Eeaction ( Cystein).— Treat the hydrochloric
acid solution with a few drops dilute ferric chloride solution
and then ammonia. The liquid will become beautifully red,
darkening on shaking with air.
AXENFELD'S Test (Albumin in Urine). — Acidify with formic
acid and add, drop by drop, a 0.1 per cent gold chloride solu-
tion and warm. The solution becomes first red, then purple
red, and, on the further addition of gold chloride, blue, and
lastly, a blue precipitate is produced. The red coloration is
characteristic of albumin, while the blue and violet may be
produced by other bodies, such as glucose, glycogen, starch,
leucin, tyrosin, uric acid, urea, creatinin, etc.
BAEYER'S Reaction (Glucose).— On boiling a glucose solu-
tion with ortho-nitrophenyl propiolic acid and sodium carbo-
nate indigo is formed. With an excess of glucose this blue,
is converted into indigo white,
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 6?
BAEYER'S Reaction (Lidol). — A watery solution of indol
gives with fuming nitric acid a red liquid and then a red
precipitate of nitroso-indol nitrate, C16H13(NO)N21HN03. It
is better to first add two or three drops of nitric acid and
then a 2$ solution of potassium nitrite, drop by drop. (Sal-
kowski.)
BARFOED'S Reagent (Dextrose). — Dissolve 1 part copper ace-
t:ite in 15 parts water; to 200 c.c. of this solution add 5 c.c.
of acetic acid containing 38 per cent of glacial acetic acid.
On heating this reagent with a dextrose solution a reduction
of copper suboxide is produced, but not when heated with
lactose or maltose.
BAUMANN'S Reaction (Dextrose).— If a watery solution of
grape sugar is treated with benzoyl chloride and an excess of
caustic soda, and shaken until the odor of benzoyl chloride
has disappeared, a precipitate of benzoic acid ester of dextrose
will be produced, which is insoluble in water or alkalies.
BAUMANN and GOLDMAN'S Test (Cystin).— Shake the solu-
tion of cystin in caustic soda with benzoyl-chloride; a volumi-
nous precipitate of benzoyl cystin is produced. The sodium
salt precipitates as silky plates, which are readily soluble in
water, but nearly insoluble in an excess of caustic soda.
BERTHELOT'S Test (Phenol). — On adding sodium hypo-
chlorite to an ammoniacal solution of phenol a beautiful blue
coloration is produced.
BILE ACIDS. See Drechsel, Mylius, Pettenkofer, 8trassburg,
Udransky.
BILE PIGMENTS. See Capranica, Dragendorff, Dumontpal-
lier, Fleischl, Gmelin, Huppert, Jolles, Le Nobel, Mareclial,
Rosenbach; Stokvis, Smith) Trousseau, Uttzmann, Vitalli,
68 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
BILIRUBIN. See Ehrlich.
BIURET Reaction (Proteids). See PIOTROWSKI'S Reaction.
BIURET Reaction ( Urea). — Heat urea in a dry test tube until
completely molten ; continue the heat for some time. When
cold, dissolve in water, add abundant caustic soda and a dilute
solution of copper sulphate, drop by drop. The solution be-
comes first pink, then reddish violet, and lastly bluish violet,
the more copper sulphate solution is added.
BLOOD. See Almen, Hoppe-Seyler, Hayem, Robert, Laden-
dorff, Pacini, Rubner, Salkowski, Struve, Wetzel, v. ZalesJci.
BOAS'S Test (HCl in Contents of Stomach). — Dissolve 5
grms. pure resorcin and 5 grms. white sugar in 100 grms. di-
lute alcohol. A few drops of this reagent are spread out in a
thin layer upon a porcelain dish, and then gently heated. On
allowing a drop of the filtrate from the stomach to flow across
it, or a glass rod dipped in the solution touched to it, a deep
scarlet streak is developed. If the solution is very dilute, no
change is observed until the solution evaporates entirely to
dryness.
BOEDEKER'S Reaction (Albumin). — Acidify the liquid with
acetic acid and add a solution of potassium ferrocyanide, drop
by drop. White precipitate of albumin will be formed.
BOTTGER'S Test (Dextrose). — Make the liquid alkaline with
carbonate of soda or potash, add some solid bismuth subni-
trate and boil. The presence of dextrose is shown by the
darkening of the bismuth salt or a black precipitate.
BRAUN'S Reaction (Glucose). — Warm the glucose solution
with caustic soda or potash until it is yellow ; now drop into
this a dilute solution of picric acid, and heat to boiling. A
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 69
deep red coloration will be the result. Creatinin gives this
same reaction even in the cold, also acetone, though faintly.
BRUCKE'S Reagent (Proteids). — Saturate a boiling 10 per
cent solution of potassium iodide with freshly precipitated
mercuric iodide; on cooling this is filtered, and the filtrate
employed with hydrochloric acid as a precipitant for the pro-
teids.
CAPRAXICA'S Reactions (Guaniri). — 1. A warm solution of
guanin hydrochloride with a cold saturated solution of picric
acid gives a yellow precipitate, consisting of silky needles.
2. With a concentrated solution of potassium chromate
guanin solutions give an orange-red crystalline precipitate,
very insoluble in water.
3. On the addition of a concentrated solution of potassium
ferricyanide to a guanin solution a prismatic, yellowish-brown
precipitate is formed.
CAPRANICA'S Reaction (Bile Pigments). — Shake the solution
with chloroform containing some bromine ; it becomes first
green, indigo blue, violet, yellowish red, and lastly colorless.
If the green or blue solution is shaken with HC1, the color is
taken up by the acid.
CELLULOSE. See Sclmlze, Schweitzer.
CHAUTARD'S Test (Acetone). — Pass sulphurous acid through
a solution of 0.25 grms. fuchsin in 500 c.c. water until the
solution is yellow in color. Add to a portion of this the liquid
to be tested for acetone. If present, the liquid will be colored
violet.
CHOLESTERIN. See Liebermann, and Burchard, Obermul-
ler, Salkowski, Scliiff, Scliidze, Zwenger.
70 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
Cholesterin. — 1. The crystal is treated with a mixture of 5
parts sulphuric acid and 1 part water, when colored rings are
produced, first a bright carmine red and then violet.
2. On the addition of a little iodine solution to the above
the crystals will be colored variously — blue, red, green, violet.
3. If a trace of cholesterin is gradually heated to dryness
with a few drops of nitric acid, a yellow spot is produced,
which turns red on the addition of ammonia. This red
color is not changed by the addition of caustic soda, thus dif-
fering from the murexid test for uric acid.
CIAMICIAN and MAGNANINI'S Test (Skatol). — On warming
skatol with sulphuric acid a beautiful purple-red coloration is
produced.
COHEN'S Test (Albumin). — Add a solution of potassium
iodide and potassium bismuthic iodide to the acid solution of
albumin. Precipitation of the albumin occurs. Alkaloids
are also precipitated.
CREATININ. See Jaffe, Kerner, v. MascJike, Salkowski,
Weyl.
CRISMER'S Test (Glucose). — A solution of 1 part safranine
in 1000 parts water is decolorized or yields a pale yellow color
when heated to boiling with an alkaline solution of glucose.
Safranine solution is not decolorized when heated in alkaline
solution with uric acid, creatin, or creatinin.
CYSTEIN. See AndreascTi.
CYSTIN. See Baumann and Goldmann, Liebig, Milller.
DAVY'S Test (Phenol). — Add 3-4 drops molybdic-sulphuric
acid (a solution of 1 part molybdic acid in 10 or more parts
cone, sulphuric acid) to 1-2 drops of the phenol solution. A
pale yellow or yellowish-brown coloration is the result, which
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 71
passes to a chestnut or reddish brown and then to a beautiful
purple. Gentle heat facilitates the reaction.
DEXIGES'S Test (Uric Acid}. — If uric acid is converted into
alloxan by the careful action of nitric acid, and the excess of
nitric acid expelled by gentle warming and then treated with
a few drops sulphuric acid and also a few drops commercial
benzol (containing thiophen),ablue coloration is produced.
DEXTROSE. See Glucose.
DIETRICH'S Reaction (Uric Acid). — Add a solution of
sodium hypochlorite or hypobromite to the uric acid solu-
tion, when a red coloration is produced. This coloration
disappears on adding caustic alkali.
DONNE'S Test (Pus). — Stir a small piece of caustic potash
with the mass to be tested. If pus is present, the mass is con-
verted into a slimy tough material.
DRAGENDORFF'S Test (Bile Pigments). — Place a few drops
of the urine on an unglazed porcelain surface and when it has
been absorbed add a drop or two of nitric acid. Several
rings of color will be produced if bile is present, chief
amongst these rings being the green ring, which is char-
acteristic of bile pigments.
DRECHSEL'S Test (Bile Acids). — Treat the substance with a
little cane sugar and a few drops of a mixture of 5 vols.
syrupy phosphoric acid and 1 vol. water. Warm on water-
bath, when a beautiful red coloration is produced if bile acids
are present.
DUMONTPALLIER'S Test (Bile Pigments). See SMITH'S Test.
EIJKMAX'S Test (Phenol). — Mix the phenol solution with a
few drops of an alcoholic solution of nitrous acid ethyl ether
72 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
and an equal volume concentrated sulphuric acid. A red
coloration is the result.
EISELT'S Reaction (Melanin in Urine). — Urine containing
melanin becomes dark-colored with oxidizing reagents, such
as concentrated nitric acid, potassium dichromate, and sulphu-
ric acid, as well as with free sulphuric acid.
EHRLICH'S Reaction (BiliruUn). — To a solution of biliru-
bin in chloroform add an equal volume or twice its volume of
a solution of sulpho-diazobenzol (1 grm. sulphanilic acid, 15
c.c. of hydrochloric acid, and 0.1 grm. sodium nitrite, diluted
to 1 litre with water). Then add as much alcohol as is
needed to render the solution clear. The liquid, which is of
a yellow color at first, assumes a beautiful red tint. On add-
ing HC1, drop by drop, the color changes first to violet and
then to an intense blue. On now carefully pouring into the
test-tube a solution of potassium or sodium hydrate three
zones of color are visible: near the alkaline solution, where
the reaction begins, the color is green; at the surface, where
the reaction is still acid, the original blue tint persists;
whilst intermediate betweent hese two zones is a red, neutral
zone.
EWALD'S Test (Hydrochloric Acid in contents of stomach).
— Dilute 2 c.c. of a 10$ solution of potassium sulpho-cyanide
and 0.5 c.c. of a neutral solution of iron acetate to 10 c.c.
with water. Place a few drops of this ruby-red solution in a
porcelain dish and allow 1-2 drops of the liquid to be tested
to flow gently thereon. In the presence of HC1 a faint violet
cloud is observed where the two liquids come in contact with
each other. On mixing the color becomes mahogany brown.
Peptones or salts do not interfere with this reaction.
FEELING'S Reagent (Glucose). — 1. Dissolve 34.65 grammes
pure copper sulphate in 1000 c.c. water.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 73
2. Dissolve 173 grammes Rochelle salts in 350 c.c. water,
adding 600 c.c. of a caustic soda solution of a specific gravity
of 1.12, and dilute to 1000 c.c. with water. For use mix equal
parts of the above solutions and dilute with an equal volume
of water.
Fehl ing's Solution.
10 c.c. — 0.0500 gramme dextrose, laevulose, or invert sugar.
10 c.c. — 0.0475 gramme cane-sugar (after inversion).
10 c.c. = 0.07143 gramme milk-sugar (lactose).
10 c.c. = 0.0807 gramme malt-sugar (maltose).
FLEISCHL'S Reaction (Bile Pigments). — Treat the urine
with a concentrated solution of sodium nitrate and add con-
centrated sulphuric acid by means of a pipette. This latter
sinks to the bottom of the test-tube and produces the colora-
tion, as in Gmelin's test.
FROHDE'S Reaction (Proteids). — On heating a solid proteid
with sulphuric acid containing molybdic acid a beautiful dark
blue color is produced.
FROHN'S Reagent. — Treat 1.5 grammes freshly precipitated
bismuth subnitrate with 20 c.c water, heat to boiling, and
then add 7 grammes potassium iodide and 10 c.c. hydrochloric
acid.
FURBRINGER'S Reagent (Albumin). — Gelatin capsules con-
taining the double salt of mercuric chloride and sodium chlo-
ride with citric acid.
FURFUROL Reaction (Proteids). — On heating proteids with
sulphuric acid furfurol is produced, which may be detected
by various means (see MOLISCH, SCHULTZE.)
GALLOIS'S Test (Inpsit). — Evaporate the inosit solution to
incipient dryness and moisten the residue with a little mer-
curic nitrate solution, when a yellowish residue is obtained on
74 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
drying. This yellow color becomes beautifully red on strongly
heating, but disappears on cooling, but reappears on gently
heating again.
GENTELE'S Test (Glucose). — If a glucose solution is added
to a solution of potassium ferricyanide made alkaline with
caustic soda or potash, it is decolorized with the formation of
potassium ferrocyanide on gently warming. Uric acid also
gives this same reaction even in the cold.
GERHARDT'S Reaction (Aceto-acetic Acid). — Aceto-acetic
acid gives a wine-red coloration with a dilute, not too acid,
ferric chloride solution. In testing urine treat 1-15 c.c. with
ferric chloride as long as it gives a precipitate, filter the pre-
cipitate of ferric phosphate, and add some more ferric chloride
to the filtrate. In the presence of the acid a claret-red color
is produced. The urine may also be acidified with sulphuric
acid and shaken with ether (which takes up the acid). Now
shake the removed ether with a very dilute watery solution of
ferric chloride and the watery layer becomes violet red or
claret red. The color disappears on warming.
GERHARDT/S Test (Uroliliri). — Extract the urobilin from the
solution by shaking with chloroform. Treat this chloroform
extract with iodine solution and then a solution of caustic
potash, when a beautiful green fluorescence is the result.
GERRARD'S Test (Glucose). — Add a 5$ solution of potassium
cyanide to Fehling's solution until the blue color just begins
to disappear. On heating this solution to boiling with a glu-
cose solution no precipitation of cuprous oxide is produced,
but the solution will be decolorized more or less.
GLOBULIN. See Hammarsten, Polil.
GLUCOSE. See Almen, Barfoed, Bdttger, Baumann, Baeyer,
Braun, Crismer, Felding, Gentele, Gerrard, Haines, v.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 75
Jakscli, Knapp, Lowentlial, Moore, Moliscli, Mulder, Nylan-
der, Penzoldt, Pavy, Roberts, Rubner, Soldani, Schiff, Tram-
mer, Wenders.
GMELIN'S Test (Bile Pigments). — If nitric acid containing
some nitrous acid is carefully poured beneath a solution con-
taining bile pigments, a series of colored layers are obtained at
the juncture of the two liquids in the following order from
above downwards : green, blue, violet, red, and reddish yel-
low. The green ring must never be absent, and also the
reddish violet must be present at the same time, otherwise
the reaction may be confused with that for lutein, which gives
a blue or greenish ring. The nitric acid must not contain too
much nitrous acid, for then the reaction takes place too
rapidly and does not become typical. Alcohol must not be
present, because it gives a play of colors with the acid.
GRIESS'S Reagent (Nitrous Acid). — A solution of metadiam-
ido-benzol (melting at 63° C.) gives an intense yellow colora-
tion with dilute solutions containing nitrous acid which have
been acidified with a few drops sulphuric acid.
GRIGG'S Test (Proteids). — A solution of meta-phosphoric
acid gives a precipitate with all proteids with the exception
of the peptones.
GUANIN. See Capranica.
Test (Acetone). — Add an alcoholic solution of
iodine to the liquid to be tested and then ammonia. On
standing a precipitate of iodoform and a black precipitate
of iodide of nitrogen is formed, but this latter gradually dis-
appears on standing, leaving the iodoform visible.
GUNZBURG'S Eeagent (Hydrochloric Acid). — Dissolve 2
grms. phloroglucin and 1 grm. vanillin in 100 c.c. alcohol.
In testing for the presence of free HC1 add an equal amount of
76 HANDBOOK FOR BIO-CIIEMICAL LABORATORY.
the above reagent to the liquid to be tested in a porcelain dish
and evaporate the mixture on the water-bath. In the pres-
ence of HC1 a delicate rose-red coloration is observed in the
residue in the porcelain dish.
See Teichmann.
HAEMOGLOBIN. See Kobert.
HAINES'S Solution (Glucose). — Dissolve 30 grains pure cop-
per sulphate in -J- ounce distilled water and add £ ounce pure
glycerin, mix thoroughly and add 5 ounces liquor of potassae.
HAMMARSTEN'S Test (Globulin). — Add powdered magne-
sium sulphate to the neutral solution until no more of the
salt dissolves. The globulin will be thus precipitated and
separated by filtration and washed with a saturated solution
of magnesium sulphate.
HAYEM'S Solution (Blood). — This solution is prepared by
dissolving 1 grm. sodium chloride, 5 grms. sodium sulphate,
0.5 grm. mercuric chloride, in 200 c.c. distilled water. It is
used in the microscopical examination of the form elements
of the blood.
HELLER'S Test (Albumin).— Float the liquid to be tested
on the surface of nitric acid. The presence of albumin is
shown by a well-defined white ring between the two liquids.
With this test even 0.02 p.m. albumin may be detected with-
out difficulty.
HERZBERG'S Eeagent (Free Inorganic Acids). — Paper
moistened with a solution of Congo red and dried turns bluish.
black or blue when moistened with hydrochloric acid. The
delicacy of. this reaction is diminished by the presence of pro-
teids or salts in large quantities.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 77
HEYNSIUS'S Test (Albumin).— Strongly acidify the solution
with acetic acid and add a few cubic centimetres of a satu-
rated solution of sodium chloride and boil. In the presence
of albumin a flocculent precipitate is produced.
Test (Albumin). — Add solid meta-phos-
phoric acid to the liquid to be tested, when a cloudiness or
precipitate is formed if albumin is present.
HIPPURIC ACID. See Lilcke.
HOFMANX'S Test (Ty rosin). — Add a few drops Millon's
reagent to the solution to be tested and boil for a time. In
the presence of tyrosin the liquid becomes a beautiful red and
then yields a red precipitate. The test may also be applied
by first adding mercuric nitrate and boiling, and after this
adding nitric acid containing some nitrous acid.
HOFMEISTER'S Test (Peptones). — A solution entirely free
from albumin gives a precipitate with an acetic acid solution
of phospho-tungstic acid. The phospho-tungstic acid may
be prepared by dissolving commercial sodium tungstate in
hot water and adding phosphoric acid until acid in reaction.
This liquid is strongly acidified with hydrochloric acid after
cooling and filtered after 24 hours.
HOFMEISTER'S Test (Leucin).—0n warming a solution of
leucin with mercurous nitrate a deposit of metallic mercury is
formed,
HOPPE SEYLER'sTest (Carbon Monoxide in Blood.) — Treat
the blood with double its volume of caustic soda solution of
1.3 sp. gr. Ordinary blood is converted into a dingy brown-
ish mass, which when spread out on porcelain is brown, with
a shade of green. Carbon monoxide blood gives under the
same conditions a red mass, which if spread out on porcelain
shows a beautiful red color.
78 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
HOPPE SEYLER'S Test (Xanthin). — Add some chloride of
lime to some caustic soda in a porcelain dish and add the
xanthin to this mixture; at first a dark green and then quick-
ly a brownish halo forms around the xanthin-grains and then
disappears.
HUPPERT'S Reaction (Bile Pigments). — Treat the solution
with milk of lime or with a solution of calcium chloride and
then precipitate with ammonia. This precipitate, containing
bilirubin calcium, is filtered, washed with water, transferred
while moist to a test-tube and treated with alcohol which has
been acidified with sulphuric acid, and heated to boiling for
some time, when the liquid becomes emerald green or bluish
green in color.
HYDROCHLORIC ACID IN CONTENTS OF STOMACH. See Boas,
Ewald, Gunzburg, Herzberg, v. Jaksch, Molir, Maly, Rabu-
teau, Reocli, Szabo, Uffelmann, v. Velden, Witz.
HYDROGEN PEROXIDE. See Wurster.
HYPOXANTHIN. See Kossel.
INDICAN. See Jaffe, MacMunn, Obermeyer, Weber.
INDIGO RED. See Rosenbach, Rosin.
INDOL. See Baeyer, Nmcki, Salkowski.
INOSIT. See Gallois, Scherer, Seidel.
JACQUEMIN'S Test (Phenol). — Treat the solution with an
equal volume of anilin and then a solution of sodium hypo-
chlorite, when a blue coloration is the result. Acids turn the
liquid red and alkalies turn it blue again. .
JAFFE'S Test (Indican). — Treat 20 c.c. of the solution to bo
tested with an equal volume of hydrochloric acid and add, by
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 79
means of a pipette, small amounts of a concentrated solution
of chloride of lime or % per cent potassium permanganate
solution, drop by drop, and after each drop shake the mixture.
In the presence of indican the mixture turns blue, due to the
production of indigo blue. An excess of oxidizing reagent,
especially chloride of lime, interferes with the reaction, and
must therefore be avoided. If 2-3 c.c. of chloroform are
shaken with the blue solution, it will be colored blue by the
indigo blue formed.
JAFFE'S Keaction (Crcatinin).— Treat the solution with a
rather concentrated watery solution of picric acid and a few
drops of caustic potash solution. In the presence of creatinin
a red coloration, lasting several hours, is produced on warm-
ing. This color changes to yellow on the addition of acid.
Aceton and glucose give a similar reaction.
v. JAKSCH'S Test (Glucose). — Add in a test-tube containing
8-10 c.c. of the solution to be tested two knife-points of
phenyl hydrazin hydrochloride and three knife-points of
sodium acetate, and when the added salts do not dissolve on
warming, add more water. The mixture is heated in boiling
water for one hour. It is then poured into a beaker-glass of
cold water. In the presence of glucose a precipitate consist-
ing of groups of yellow needles of phenyl glucosazone is
formed. In doubtful cases determine the melting-point of
these yellow crystals to be 204-205° C.
y. JAKSCH'S Test (Melanin). — Add a few drops of a concen-
trated solution of ferric chloride to the liquid to be tested. In
the presence of melanin it turns gray, and on the addition
of more ferric chloride the precipitate, consisting of the color-
ing matter and the phosphates, is redissolved.
Y. JAKSCH'S Test (IIC1 in Contents of Stomach).— Paper moist-
ened with a saturated, watery solution, of benzQ^wrpurin 6 B»
80 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
and dried gives with dilute solutions of HC1 a beautiful
violet coloration. If the paper becomes dark blue, the solu-
tion contains more than 0.4 grin. HC1 in 100 c.c. of the
solution.
v. jAKSCH'sTest (Uric Acid).— This consists in substituting
chlorine or bromine water or nitrous acid for the nitric acid
in the murexid test (see Murexid Test.) This reaction differ-
entiates between uric acid and the xanthin bases.
•
JOHNSON'S Test (Albumin). — Float the acidified solution
on a cold saturated solution of picric acid. If albumin is
present, a precipitation of the albumin occurs between the two
liquids.
JOLLES'S Test (Bile Pigments in Urine). — Place 50 c.c. of
the urine in a stopper cylinder, add a few drops of 10$ hydro-
chloric acid and an excess of a barium chloride solution with
5 c.c. chloroform, and shake for several minutes. After 10
minutes remove the chloroform and the precipitate by means
of a pipette and place in a test-tube and heat on the water-
bath to about 80° 0. After the evaporation of the chloro-
form decant the liquid from the precipitate carefully and
allow 3 drops concentrated sulphuric acid containing^ fuming
nitric acid to flow down the sides of the test-tube. In the
presence of bile pigments the characteristic coloration is pro-
duced.
KERNER'S Reaction (Creatiniri). — A solution of creatinin
acidified with a mineral acid gives a crystalline precipitate
with phospho-tungstic or phospho-molybdic acids, even in
very dilute solutions.
KNAPP'S Solution (Glucose). — Dissolve 10 grms. chemically
pure dry mercuric cyanide in 100 c.c. caustic soda solution
of a specific gravity of 1-145 and dilute to 1 litre. On heating
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 81
a glucose solution with the above solution diluted with water
a reduction of metallic mercury takes place. Ten c.c. of this
solution are reduced by 0.025 grm. glucose.
ROBERT'S Test (Hcemoglobin). — Shake the solution with
zinc powder or treat with a solution of zinc sulphate or acetate,
when a precipitate of zinc haemoglobin is formed. This pre-
cipitate when collected is colored red by alkalies.
KOSSEL'S Test (Hypoxantliin). — Treat the solution with
zinc and hydrochloric acid and then make alkaline with
caustic soda or potash. In the presence of hypoxanthin the
solution becomes first ruby red and then brownish red in
color.
LACTIC ACID. See Uffelmann.
LADEXDORFF'S Test (Blood). — Treat the liquid with tinct-
ure of guaiacum and then with oil of eucalyptus, when the
lower layer becomes blue and the upper layer violet if blood
is present.
LAXDOLT'S Test (Phenol). — On treating the solution with
bromiue water a white crystalline precipitate of tribrom-
phenol (C6H2,Br3,OH) is produced.
LANG'S Reaction (Taurin). — On boiling a solution of
taurin with freshly precipitated mercuric oxide a white com-
bination occurs which appears as a precipitate.
LEGAL'S Test (Acetone). — Treat the acetone solution with a
few drops of a freshly prepared solution of sodium nitro-
prusside and then with caustic potash or soda solution. The
solution becomes ruby red in color, but if saturated with
acetic acid the color becomes carmine or purplish rod.
Oeatinin gives the ruby-red color, with sodium nitro-prusside
82 HANDBOOK FOR BIO-CHEMICAL LABORATORY,
and alkali, but this turns yellow, and then gradually green
and blue, when saturated with acetic acid. Ammonia may
be substituted for the caustic soda or potash and gives the
same reaction with acetone, but no reaction with creatinin.
LE NOBEL'S Modification of LEGAL'S Test (Acetone).—
Instead of using caustic potash or soda with the sodium nitro-
prusside he suggests the use of ammonia, which produces a
ruby-red reaction with acetone, but not with creatinin. (See
Legal's Test.)
LE NOBEL'S Test (Bile Pigments). — Treat the liquid with
zinc chloride and a few drops of tincture of iodine. A di-
chroitic play of colors is the result.
LEUCIN. See Hoffmeister, Scherer.
LIEBEN'S Test (Acetone). — When a watery solution of
acetone is treated with alkali and then a solution of iodine in
potassium iodide solution and gently warmed, a yellow pre-
cipitate of iodoforrn is formed, which is known by its odor and
by the appearance of the crystals (six-sided plates or stars)
under the microscope.
LiEBERMAN^-BuRCHARD's Test (Cholesterin). — Dissolve
the substance in acetic anhydride and then add concentrated
sulphuric acid, when a beautiful violet color is produced, and
this passes quickly to green if cholesterin is present.
LIEBERMAN^S Test (Proteids).— Treat the proteid, pre-
viously washed with alcohol and ether, with concentrated
fuming hydrochloric acid, when a beautiful violet-blue colora-
tion is the result. This liquid gives an absorption-band be-
tween E and b.
LIEBIG?S Test (Cystin). — Boil the substance with caujtic
alkali containing lead oxide. If cystin is present, a pre-
cipitate of black lead sulphide is produced.
HANDBOOK FOB BIO-CHEMICAL LABORATORY. 83
LOWENTHAL'S Test (Glucose). — On boiling a glucose solution
witli a solution of ferric chloride dissolved in tartaric acid and
sodium carbonate it darkens and soon deposits a voluminous
precipitate of iron oxide. This test cannot be applied to the
urine, as all urines give it,
LUCRE'S Reaction (Hippuric Acid). — Evaporate the sub-
stance to dryness with nitric acid, when an intense odor of
nitro-beuzol (oil of bitter almonds) is generated when the
residue is heated.
NN'S Test (Indican in Urine). — Equal parts of urine
and hydrochloric acid and a few drops nitric acid are boiled,
cooled, and agitated with chloroform. The chloroform is
colored violet and shows an absorption-band before D, due to
indigo blue, and another after D, due to indigo red.
MACWILLIAM'S Test (Albumin). — Add a concentrated wa-
tery solution of salieyl sulphonic acid to the acid-reacting
solution, when a cloudiness or precipitate will be formed in
the presence of albumin. In the presence of peptones or
albumoses the precipitate disappears on boiling, but reap-
pears on cooling.
MALERBA'S Test (Acetone). — A solution of dimethylpara-
phenylendiamine gives a red coloration with acetone, which
gives an absorption spectrum very similar to oxyhaemoglobin.
MALY'S Test (HCl in Contents of Stomach). — Place liquid
to be tested in a glass dish and add as much ultramarine to
make it just blue. Then cover the dish with a watch-glass
after having suspended a piece of leud-paper in the upper
part of the dish. On warming the mixture on the water-
bath after 15 minutes in the presence of HCl the blue color
of the mixture has changed to brown and the lead-paper will
have turned dark, due to the development of H,S.
84 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
Zinc sulphide may be substituted for the ultramarine,
using a knife-point of the powder to 20 c.c. of the filtered
contents of the stomach.
MANDEL'S Test (Proteids). — A 5 per cent solution of chro-
mic acid produces a precipitate with solutions of proteids.
If the solution is first made acid with acetic or citric acid, the
precipitate produced is flocculent and settles rapidly. It
produces a marked cloudiness with 1 part albumin dissolved
in 50,000 parts water. Chromic acid solution may be substi-
tuted for nitric acid in Heller's test, using a 10 per cent
solution.
MARECHAL (Bile Pigments). See SMITH'S Test.
v. MASCHKE'S Reaction (Creatinin). — Dissolve the creatinin
in a cold saturated solution of sodium carbonate and add a
a few drops of Fehling's solution. An amorphous flocculent
precipitate is obtained in the cold, but better on warming
to 50-60° C.
MEHU'S Test (Albumin). — Shake 100 vols. of the solution
with 2-3 vols. nitric acid and 10 vols. of a solution of 1 part
phenol and 1 part acetic acid in 2 parts 90$ alcohol. In the
presence of albumin a precipitate is produced. Instead of
nitric acid one half a volume of a saturated solution of
sodium sulphate may be used.
MELANIN. See Eiselt, v. Jakscli, ThormdJikn, Zeller.
MICHAILOW'S Test (Proteids).— Treat the solution with
ferrous sulphate, and allow concentrated sulphuric acid to
flow under the solution, and then add carefully very little
nitric acid. Besides a brown ring, a blood-red coloration will
also be produced,
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 85
MILLON'S Reagent (Proteids). — Dissolve 1 part mercury in
2 parts nitric acid (sp. gr. 1.42), allow to stand some time,
and then apply heat. After complete solution of the mer-
cury add 1 voh of this solution to 2 vols. of water. Allow
to stand a few hours and decant the supernatant liquid.
This reagent gives with solutions of proteid bodies a precipi-
tate which slowly at the ordinary temperature, but quickly at the
boiling-point, turns red, depending upon the amount of albu-
min. Solid albuminous bodies give the same reaction. This
reaction depends on the presence of the aromatic group in
the proteid, and is also given by tyrosin and other benzol
derivatives with a hydroxyl group in the benzol nucleus.
MOHR'S Test (HClin Contents of Stomach). — A solution of
iron acetate (free from alkali acetates) so diluted as to have
only a light yellow color is treated with a few drops of a solu-
tion of potassium sulpho-cyanide. No change of color should
be produced, but if the filtered contents of the stomach are
added, and they contain free HC1, an intense red coloration
is the result. This color disappears on the addition of sodium
acetate.
MOLISCH'S Test (Glucose). — 1. Treat £ to 1 c.c. of the solu-
tion with 2 drops of a 15-20$ alcoholic solution of a naph-
thol. The liquid becomes cloudy, due to the precipitation of
some of the naphthol, but on the addition of 1-2 c.c. concen-
trated sulphuric acid a beautiful deep violet coloration is pro-
duced, which forms a violet precipitate on diluting with
water.
2. Instead of employing a solution of a naphthol he also
suggests the use of a 15-20$ alcoholic solution of thymol,
applied as above. In the presence of glucose it is colored
ruby red and becomes carmine red on dilution with water.
MOORE'S Test ( Glucose).— If a glucose solution is treated with
about i of its volume of caustic soda or potash and warmed, the
86 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
solution becomes first yellow, then orange, yellowish brown,
and lastly brown, depending upon the amount of glucose
present. A faint odor of caramel is also observed, and this
is more pronounced if the solution is acidified.
MULDER'S Test (Glucose). — Treat the solution with a solu-
tion of sodium carbonate and add a solution of indigo car-
mine. On heating in the presence of glucose the solution
becomes decolorized, and turns blue again on shaking with
air.
MULDER'S Test, also Xantho-proteic Reaction (Proteids). —
On treating proteids with concentrated nitric acid they are
colored yellow. On adding ammonia or cavstic soda or potash
they turn orange yellow.
MULLER'S Test (Cystin). — Dissolve the cystin by boiling
with caustic potash, dilute with water when cold, and add a
solution of sodium nitro-prusside, when a violet coloration is
produced. This color changes rapidly to yellow.
MUREXID Test (Uric Acid). — Heat the powder gently on a
watch-glass with a drop or two of strong nitric acid. A red
residue is produced, which, when cold, turns a purple red
when ammonia is added (purpurate of ammonium). When
caustic soda or potash is added to this, it becomes more blue
or bluish violet. Better results are obtained if the heating is
done over the water-bath, and not over a naked flame.
MYLIUS'S Modification of Pettenkofer's Test (Bile Acids).—
To each cubic centimetre of the alcoholic solution of bile
acids add 1 drop of furfurol solution and 1 c.c. concentrated
sulphuric acid, and cool when necessary, so that the test does
not become too warm. A red coloration is the result, and
this color does not disappear at the ordinary temperature, but
becomes more bluish violet in the course of a day.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 8<
NENCKI'S Test (Indol). — Inclol gives a pronounced red col-
oration with nitric acid containing nitrous acid. In concen-
trated solution a red precipitate may form. This reaction is
not given by skatol.
NITROUS ACID or Nitrites. See Griess.
NYLANDER'S Test (Glucose). — Dissolve 4 grms. Rochelle
salts in a solution of 10.33 grms. NaHO in 100 c.c. water.
Add to this 2 grins, bismuth subnitrate and digest on the
water-bath until as much of the bismuth salt is dissolved
as possible. On heating 10 vols. of a glucose solution with 1
vol. of the above solution for 2-5 minutes a black precipitate
or a dark coloration is the result.
OBERMEYER'S Test (Indican in Urine). — Precipitate the
urine with a lead acetate solution (1 to 5), being careful not to
add an excess of lead solution. Filter through a dry folded
filter and shake the filtrate with an equal volume of fuming
hydrochloric acid which contains 1-2 parts ferric chloride
solution to 500 parts of the acid. Continue shaking for 1 or
2 minutes, and then add some chloroform, which takes up
the indigo blue produced and is colored blue.
OBERMULLER'S Test (Cholesteriii).— Fuse the cholesterin with
2 or 3 drops propionic acid anhydride in a test-tube over a
small naked flame. On cooling the fused mass is first violet,
then blue, green, orange, carmine, and lastly copper red.
OLIVER'S Test (Albumin). — Mix equal parts of sodium
tungstate solution (1 to 4) and a saturated solution of citric
acid, (10 to 6). The urine is floated on this solution, and in
the presence of albumin a white ring is obtained at the con-
tact of the two liquids.
PACINI'S Liquid (Blood). — One part corrosive sublimate, 2
parts sodium chloride, 13 parts glycerin, 113 parts distilled
88 HANDBOOK FOE BIO-CHEMICAL LABORATORY.
water. This mixture should stand 2 months. For use mix
1 part of this solution with 3 parts water and filter.
PA YY'S Reagent (Albumin). — This is a practical dry reagent
for albumin. It consists of small disks or plates of citric acid
and sodium ferrocyanide.
PAVY'S Solution (Glucose). — Mix 120 c.c. of the ordinary
Fehling's solution with 300 c.c. of strong ammonia (sp. gr.
0.88) and with 400 c.c. more of caustic soda solution of sp. gr.
1.14. Now dilute to 1000 c.c. with water. One hundred c.c.
of this solution is reduced by glucose to the same extent as
10 c.c. of the ordinary Fehling's solution. This solution
becomes decolorized by boiling with glucose solution.
PENZOLDT'S Test (Glucose). — Dissolve 1 part diazobenzo-sul-
phonic acid in 60 parts of water, and to facilitate solution add
1 or 2 drops caustic potash.
In applying this tejst make some of the solution to be
tested strongly alkaline, and then add an equal volume of the
above solution of diazobenzol sulphonic acid. It is advisable
to do the same with a solution free from sugar. On allowing
to stand the mixture becomes yellowish red or light claret
red, then darker, and in the presence of considerable glucose
it becomes dark red and opaque. The red color has a bluish
shade.
PENZOLDT'S Test (Acetone). — A warm saturated solution of
orthonitrobenzaldehyde is treated with the liquid to be tested
for acetone and then made alkaline with caustic soda. In the
presence of acetone the liquid first becomes yellow, then green,
and lastly indigo separates, and this may be dissolved with a
blue color by shaking with chloroform.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 89
PENZOLDT and FISCHER'S Test (Phenol). — On treating a
strongly alkaline solution of phenol with a solution of diazo-
benzol sulphonic acid a deep red coloration is the result.
PEPTONES. See Hofmeister.
PETRI'S Test (Proteids). — On treating a proteid or peptone
solution with a solution of diazobenzol sulphonic acid only a
faint yellow coloration is produced, but on making the solu-
tion alkaline with caustic alkali the solution becomes orange
yellow to brown, according to concentration, and yields a red
froth on shaking.
PETTEN KOFER'S Test (Bile Acids). — A small quantity of bile
in substance is dissolved in a small porcelain dish in con-
centrated sulphuric acid and warmed, or some of the liquid
containing the bile acids is mixed with concentrated sul-
phuric acid, taking special care in both cases that the tem-
perature does not rise higher than 60-70° C. Then a 10$
solution of cane-sugar is added, drop by drop, continually
stirring with a glass rod. The presence of bile is indicated by
the production of a beautiful red liquid, whose color does not
disappear at the ordinary temperature, but becomes more blu-
ish violet in the course of a day. This red liquid shows a
spectrum with two absorption-bands, the one at F and the
other between D and E, near E.
PHENOL. See Allen, Bertlielot, Davy, Eijkmann, Jacque-
min, Landolt, Penzoldt and Fischer.
PIOTROWSKI'S Reaction (Proteids), also called Biuret Re-
action.— On heating a proteid with an excess of a concen-
trated solution of caustic soda and one or two drops of a
dilute solution of copper sulphate a violet color is produced
which deepens in tint on boiling.
90 HANDBOOK FOR BlO-CHEMlOAL LABORATORY.
PIRIA'S Test (Ty rosin}. — Dissolve the substance in con-
centrated sulphuric acid and allow to stand for -J- an hour.
Dilute with water and neutralize the solution with BaC03 and
filter. On the addition of acid-free ferric chloride to the clear
filtrate a violet color is produced in the presence of tyrosin.
The reaction is impeded by the presence of free acid.
POHI/S Test (Globulins). — He suggests to saturate the solu-
tion to one-half with ammonium sulphate, which precipitates
the globulins. Filter and wash with a one half saturated
solution of ammonium sulphate.
PROTEIDS. See Adamkiewicz, Biuret, Brilcke, Frohde,
Furfur ol, Grigg, Liebermann, Mandel, Millon, Mulder, Mi-
cliailou, Petri, Piotrowski, Reichl, Schultze, Xantho-proteic.
Pus. See Donne.
KA ABE'S Test (Albumin). — Place 1 c.c. of the liquid to be
tested in a test-tube and add a small piece of trichloracetic
acid. In the presence of albumin a white zone or ring will
be formed. The ring produced by uric acid is diffused and
not sharply defined.
RABUTEAU'S Test (HCl in Contents of Stomach). — Add
the filtered contents of the stomach to a solution containing
50 c.c. starch mucilage, 1 grm. potassium iodate, and 0.5 grm.
potassium iodide, In the presence of free HCl it will become
blue.
REES'S Test (Albumin). — An alcoholic solution of tannic
acid precipitates small amounts of albumin.
REICHL'S Test (Proteids). — Add 2-3 drops of an alcoholic
solution of benzaldehyde to the proteid solution, and then
considerable sulphuric acid which has previously been diluted
HANDBOOK FOR BIO-CHEMIC%L fc^jTRTTAEY^ 91
with an equal volume of water. Lastly, add a few drops of a
ferric sulphate solution, when a deep blue coloration will be
produced in the cold after some time or immediately on
warming. Solid proteids are also colored blue by this reac-
tion.
REOCH'S Test (Albumin). See Macwilliam.
REOCH'S Test (HCl in Contents of Stomach). — A mixture
of citrate of iron and quinine and potassium sulpho-cyanide
is colored red by the gastric juice or contents of the stomach
containing free hydrochloric acid.
REYNOLD'S Test (Acetone). — Precipitate HgO from a mer-
curic chloride solution by adding an alcoholic caustic potash
solution. To this freshly precipitated HgO add the liquid
to be tested for acetone, shake, and filter. In the presence
of acetone the filtrate contains mercury, due to the acetone
dissolving freshly precipitated HgO. The mercury is de-
tected in the filtrate by means of ammonium sulphide, which
turns black.
ROBERT'S Test (Glucose in Urine). — Take the specific
gravity of the urine at a known temperature by means of a
urinometer or py kilometer supplied with a thermometer.
Now acidify slightly with tartaric acid and add a piece of
yeast the size of a pea and shake. Allow to stand at the
temperature of the room, or, better, at 20-25° C., for 24-48
hours. The fermentation by this time will be finished. Now
filter through a dry filter and cool to the same temperature
as you took the specific gravity before fermentation. Now
take the specific gravity again.
Each degree of specific gravity lost represents 1 grain of
glucose to the ounce of urine, or if the number of degrees
lost in specific gravity is multiplied by the factor 0.23 we
92 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
obtain the percentage of glucose or grammes per 100 c.c. of
urine.
ROBERT'S Test (Albumin). — Allow the urine to flow on the
surface of a saturated common salt solution containing 5$
HC1 of specific gravity 1.052. In the presence of albumin a
white ring or zone will form between the two liquids.
He also suggests a mixture of 1 part strong nitric acid and
5 parts saturated magnesium sulphate solution. It is to be
applied as above,
ROSENBACH'S Modification of GMELIN'S Test (Bile Pig-
ments).— Filter the liquid through a very small filter. When
all liquid has passed through, apply to the inside of the filter
a drop of nitric acid which contains only very little nitrous
acid. A pale yellow spot will be formed, which is surrounded
by colored rings which are yellowish red, violet, blue, and
green.
ROSENBACH'S Test (Indigo Red or Indirubiri). — On boiling
the liquid with nitric acid indigo blue is formed from the
indigo red.
ROSIN'S Test (Indigo Red or Indirubin). — Make the liquid
alkaline with sodium carbonate and extract with ether, which
is colored red by the indigo red.
RUBNER'S Test (Carbon Monoxide in Blood). — Shake the
blood for one minute with 4-5 volumes lead acetate solution.
If the blood contains CO, it will retain its bright red color,
while if it does not it will turn chocolate brown.
RUBBER'S Test (Glucose). — Treat the liquid with an excess
of lead acetate, filter, and add ammonia to the filtrate until
no further precipitate is produced. Warm gently, when the
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 93
precipitate produced by the ammonia will be gradually col-
ored pink. This coloration diminishes on standing.
SALKOWSKI'S Keaction (Cholesteriii). — Dissolve the sub-
stance in chloroform and then treat with an equal volume of
concentrated sulphuric acid. The cholesterin soiution be-
comes first bluish red, then gradually more violet red, while
the sulphuric acid appears dark red with a greenish fluores-
cence. If the chloroform solution is poured into a porcelain
dish, it becomes violet, then green, and finally yellow.
SALKOWSKI'S Modification of HOPPE SEYLER'S Test (CO
in Blood). — Dilute the blood to be tested with 20 vols. water
and add thereto an equal volume of a caustic soda solution
of sp. gr. 1.34. If the blood contains carbon monoxide, the
mixture will become milky in a few moments and then
bright red. On standing red flakes form, which collect on
the surface of the liquid. Normal blood treated in this way
gives a dirty brown coloration.
SALKOWSKI'S Reaction (Creatinin). — If a few drops of a
freshly prepared very dilute solution of sodium nitro-prusside
are added to a dilute creatinin solution and then a few drops
of caustic soda, a ruby-red liquid is obtained which quickly
turns yellow (Weyl's reaction). If this yellow solution is
treated with an excess of acetic acid and heated, the solution
becomes first green and then blue, and finally a precipitate of
Prussian blue is obtained.
SALKOWSKI'S Test (LidoI).^-AM a few drops nitric acid to
the indol solution and then, drop by drop, a 2$ solution of po-
tassium nitrite. In the presence of indol a red color is pro-
duced, and lastly a red precipitate of nitroso-indol nitrate.
SCHERER'S Test (Inosit}.— Evaporate the substance to dry-
ness on a platinum foil with nitric acid and treat the residue
94: HANDBOOK FOR BIO-CHEMICAL LABORATORY.
with ammonia and a drop of calcium chloride solution and
carefully re-evaporate to dryness. With inosit a rose-red
residue is obtained.
SCHERER'S Test (Leucin). — Carefully evaporate the leucin
with nitric acid on a platinum foil. No markedly colored
residue is left, but on gently warming this with a few drops
of caustic soda solution a color varying from a pale yellow to
a brown (depending on the purity of the leucin) is produced,
and on further concentrating over the flame it agglomerates
into an oily drop which rolls about on the foil.
SCHERER'S Test (Ty rosin). — Evaporate the substance care-
fully to dryness with nitric acid on a platinum foil. A beau-
tiful yellow residue (nitro-tyrosin nitrate) is obtained, which
gives a deep reddish-yellow color with caustic soda.
SCHIFF'S Test (Uric Acid). — Dissolve the substance in
sodium carbonate and add silver nitrate solution, when a re-
duction of black silver oxide is obtained. If a drop of the
solution of the substance in sodium carbonate is placed on a
piece of filter-paper which has been previously treated with
silver nitrate solution, a reduction of black silver oxide will
also be formed on the paper.
SCHIFF'S Test (Carbohydrate).— Strips of paper are dipped
in a mixture of equal volumes of glacial acetic acid and
xylidin, treated with very little alcohol, and dried. On ex-
posing such paper to the furfurol vapors produced by treating
glucose with sulphuric acid the paper will be colored red.
SCHIFF'S Reaction (Cholesterin).— Evaporate the substance
in a porcelain dish over a small flame with a few drops of a
mixture of 2-3 vols. concentrated hydrochloric or sulphuric
acid and 1 vol. of a medium solution of ferric chloride. In
HANDBOOK FOR BIO-CHEMICAL LABOBATORY, 95
the presence of cholesterin a reddish-violet residue is first ob-
tained and then a bluish violet.
SCHIFF'S Test (Urea). — Place a drop of a concentrated
watery solution of furfurol on the crystal of urea and then a
drop of hydrochloric acid of sp. gr. 1.10. A change of color
from yellow, green, blue, to purple is obtained. Allantoin
gives this same reaction, but less intense and not so quickly.
SCHROEDER'S Test ( Urea). — Place a crystal on a microscope-
slide and add a solution of bromine in chloroform. Urea will
not dissolve therein, but is decomposed with the develop-
ment of gas.
SCHULZE'S Reagent (Cellulose). — Dissolve iodine to satura-
tion in a zinc chloride solution of sp. gr. 1.8 to which 6 parts
potassium iodide has been added. Cellulose turns blue with
this reagent.
SCHULZE'S Test (Cholesterin). — Evaporate the substance to
dryness on the water-bath in a porcelain dish with nitric acid.
A yellow residue is obtained with cholesterin, which turns
yellowish red on the addition of ammonia.
SCHULTZE'S Test (Proteids). — Add a few drops of a dilute
cane-sugar solution and then concentrated sulphuric acid to
a solution of the proteid and warm the mixture to 60° C.,
when a beautiful bluish-red coloration is obtained. It is
important to keep the temperature at 60° C.
SCHWEITZER'S Reagent (Cellulose).— Sulphate of copper in
solution, to which some ammonium chloride has been added, is
precipitated with caustic soda ; the hydrated cupric oxide
thus obtained is washed and dissolved to saturation in 20$
ammonia. It may also be prepared by pouring ammonia on
copper turnings, the requisite oxidation of the copper being
96 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
effected by drawing a current of air through the fluid in
which the turnings are immersed. Cellulose is soluble in the
above reagent.
SEIDEL'S Reaction (Inosit). — Evaporate a small amount of
the substance to dryness in a platinum crucible with a little
nitric acid (sp. gr. 1.1-1.2), and treat the residue with ammo-
nia and a few drops of a solution of strontium acetate, lu
the presence of inosit a greenish coloration is observed, to-
gether with a violet precipitate.
SKATOL. See Ciamician and Magnanim.
SMITH'S Reaction (Bile Pigments). — Pour carefully over
the liquid to be tested tincture of iodine, whereby a green
ring appears between the two liquids.
SoLDAsri's Solution (Glucose). — Dissolve 15 grms. copper
carbonate in 1400 c.c. water and add 416 grms. potassium
bicarbonate. On heating a glucose solution with the above
solution a reduction of copper suboxide is obtained.
SPIEGLER'S Test (Albumin). — Remove mucin from the
solution by the addition of acetic acid, filter, and treat the
filtrate with a solution prepared by dissolving 8 grms. mer-
curic chloride, 4 grms. tartaric acid, in 200 c.c. water and
adding 20 grms. glycerin thereto. In the presence of albumin
a white ring is obtained between the two liquids.
STOKVIS'S Test (Bile Pigments).— Treat 20-30 c.c. of urine
with 5-10 c.c. of a solution of zinc acetate (1 to 5). The pre-
cipitate is washed on a small filter with water and then dis-
solved in a little ammonia. Filter, and the filtrate gives,
after standing in the air, a peculiar brownish-green color, and
shows the three absorption-bands of bilicyanin, the first between
C and D, the second at Z>, and the third between D and E,
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 97
STOKES'S Reagent (Reducing Oxylicemoglobin). — To a solu-
tion of ferrous sulphate add some citric or tartaric acid and
enough ammonia to make it alkaline.
STRASSBURG'S Test (Bile Acids). — Treat the liquid with
cane-sugar and dip a strip of filter-paper in this liquid. Dry
this carefully over a gas or alcohol flame and place a drop of
sulphuric acid thereon. In the presence of bile acids a red
coloration is produced on the paper. The liquid must be free
from albumin for this test.
STRUYE'S Test (Blood in Urine). — Treat the urine with
ammonia or caustic potash, and then add tannin and acetic
acid until the mixture has an acid reaction. In the presence
of blood a dark precipitate is formed. Filter, dry, and obtain
the characteristic hsemin crystals from the dry residue by the
addition of ammonium chloride and glacial acetic acid. (See
Teiclimann.)
SZABO'S Test (HCl in Contents of Stomach). — Equal
volumes of •£# solutions of ammonium sulpho-cyanide and of
sodic-ferric tartrate are mixed. On adding liquid containing
HCl to this solution, which is pale yellow, a brownish-red
color is produced.
TAXRET'S Test (Albumin). — Dissolve 3.32 grms. potassium
iodide and 1.35 grms. mercuric chloride (4 mols. KI to 1 mol.
HgCla) in 20 c.c. acetic acid and dilute to 60 c.c. When
this reagent is added to an albumin solution, a white precipi-
tate is produced.
TAURUS". See Lang.
TEICHMAXN'S Test (Hcemin Crystals).— Place a few parti-
cles of the dry residue on a microscope-slide, add a grain of
common salt, and cover with a cover-glass. Now add some
98 HANDBOOK FOR BIO-CHEMICAL LABORATORY.
glacial acetic acid under the cover-glass and warm gently not
to boil the liquid. In the presence of blood-coloring matters
the characteristic dark brown, long, rhombic crystals of haemin
are obtained. If no crystals appear after the first warming,
warm again, and if necessary add some more acetic acid.
THORMAHLEN-'S Test (Melanin in Urine).— AM sodium,
nitro-prusside, caustic potash, and acetic acid to the urine to
be tested, and in the presence of melanin a deep blue colora-
tion is the result.
TROMMER'S Test (Glucose). — Make the liquid strongly alka-
line with caustic soda and add a not too concentrated solution
of copper sulphate, drop by drop, until a little of the copper
hydrate formed remains undissolved on shaking. Now warm,
and in the presence of glucose a yellow reduction of hyd rated
suboxide of copper is first formed and then red suboxide sepa-
rates even below the boiling-point. If too little copper salt
has been added, the test will be yellowish brown in color, as in
Moore's test ; but if an excess of the copper salt has been
added, the excess of hydrate is converted on boiling into a
dark brown hydrate, which interferes with the test.
TROUSSEAU'S Test (Bile Pigments). See Dumontpallier,
Smith.
See Hoffmann, Piria, Scherer, Udransky,
Wurster.
UDRANSKY'S Test (Tyrosiri). — Dissolve a particle of the
substance in 1 c.c. of water and add 1 drop of a 0.5$ watery
furfurol solution and then 1 c.c. concentrated sulphuric acid.
The mixture becomes faintly red. Care should be taken not
to have the mixture get above 50° 0.
TJDRANSKY'S Test (Bile Acids). — Treat 1 c.c. of a watery or
alcoholic solution of the substance with 1 drop of a 0.1 ;/
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 99
watery solution of fnrfurol and allow 1 c.c. concentrated sul-
phuric acid to flow underneath this mixture. Care should be
taken to keep the mixture cool. In the presence of bile acids
a red coloration is obtained. The red color should have a
shade of blue (violet) to be characteristic of bile acids.
UFFELMANN'S Test (Lactic Acid in Contents of Stomach). —
Mix 10 c.c. of a 4$ carbolic acid solution and 20 c.c. water and
add a few drops ferric chloride solution, when an amethyst-
blue solution is obtained. With lactic acid this solution is
colored yellow.
UFFELMANN'S Test (HCl in Contents of Stomach). — Dip
strips of filter-paper in an amyl alcohol extract of huckle-
berries and dry. When the contents of the stomach contains
HCl, it will turn this paper pink.
ULTZMANN'S Reaction (Bile Pigments). — Treat the solution
with caustic potash and mix, and then acidify with hydro-
chloric acid. The solution becomes emerald green, due to
the formation of biliverdin.
UREA. See Biuret, Schiff, Schroeder.
URIC ACID. See Deniges, Dietrich, v. Jaksch, Murexid,
Schiff.
UROBILIN. See Gerhardt.
v. D. VELDEN'S Test (HCl in Contents of Stomach).— A
watery or alcoholic solution of Tropaeolin 00, which is yellow
in color, turns ruby red or deep brownish red with free hydro-
chloric acid. Paper moistened with the above solution may
be used for the test.
100 HANDBOOK FOB BIO-CHEMICAL LABORATORY.
VITA LLI'S Test (Bile Pigments.) — Treat the solution with
a few drops of a solution of potassium nitrite and then some
dilute sulphuric acid, when a beautiful green color will be
obtained. This green color changes to yellow after a time,
but first turns red or blue.
WEBER'S Test (Indican in Urine). — Treat 30 c.c. of the
urine with an equal volume hydrochloric acid and 1-3 drops
dilute nitric acid and heat to boiling. The solution becomes
dark, and if shaken with ether, when cold, the ether will be
colored red to violet, while a blue foam is observed on the top
of the ether.
WEIDEL'S Reaction (Xantliiri). — A little of the substance
is dissolved in fresh chlorine water containing some nitric
acid and evaporated on the water-bath to dryness. On expos-
ing the white or yellowish residue to the vapors of ammonia,
under a bell- jar, a red or purple violet color is produced.
WENDKR'S Test (Glucose). — Dissolve 1 part commercial
methylene blue in 3000 parts distilled water. On making this
solution alkaline with caustic potash and heating with a
glucose solution the blue color disappears and the solution
becomes decolorized.
WETZEL'S Test (CO in Blood).— Dilute the blood with 4
vols. water and treat with 3 vols. of a \<f> tannic acid solution.
In the presence of carbon monoxide the blood becomes car-
mine red, while normal blood gradually becomes gray.
WEYL'S Reaction (Great in in).— Add a few drops of a
freshly prepared solution of sodium nitro-prnsside to the
solution of creatinin and then a few drops of caustic soda. A
ruby-red liquid is obtained, which quickly turns yellow again.
The solution of creatinin zinc chloride may also be used.
HANDBOOK FOR BIO-CHEMICAL LABORATORY. 101
WITZ'S Test (HCl in the Contents of the Stomach). — A watery
solution of methyl-anilin violet is first rendered blue, then
green, and ultimately decolorized by dilute inorganic acids.
WURSTER'S Test (Tyrosin). — A boiling watery solution of
tyrosin is colored red when treated with \% acetic acid and a
sodium nitrite solution, drop by drop.
WURSTER'S Test (Tyrosin). — Dissolve the tyrosin in hot
water and to the hot solution add some dry chinon. The
solution becomes deep ruby red, which remains for 24 hours
and then passes to brown.
WURSTER'S Test (Hydrogen Peroxide). — Paper soaked with
a solution of tetramethylparaphenylendiamine turns blue
violet with hydrogen peroxide.
XANTHIN. See Hoppe Seyler, Weidel.
XAXTHO-PROTEIC Reaction (Proteids). See Mulder.
v. ZALESKI'S Test (Carbon Monoxide in Blood). — Mix 2 c.c.
of the blood with 2 c.c. water and 3 drops of a J saturated
copper sulphate solution. With normal blood u greenish-
brown precipitate is produced, while if the blood contains CO
a brick -red precipitate is obtained.
ZELLER'S Test (Melanin in Urine). — A urine containing
melanin gives, when treated with bromine water, a yellow pre-
cipitate, which gradually turns black.
ZOUCHLOS'S Test (Albumin). — The reagent consists of 100
parts 10$ potassium sulpho-cyanide solution and 20 parts
acetic acid. When this reagent is added, drop by drop, to a
solution of albumin, a marked cloudiness is observed.
ZWE:N:GERJS Test (Cholesterin). See Cholesterin Reactions
Xo. 1.
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